Derivatives of 4- or 5-aminosalicylic acid

ABSTRACT

The present invention provides new derivatives of 4- or 5-aminosalicylic acid, and a pharmaceutical composition containing these derivatives of 4- or 5-aminosalicylic acid as active ingredients, useful for the treatment of intestinal diseases such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) and for the prevention/treatment of colon cancer. More particularly, these derivatives comprise a hydrogen sulfide releasing moiety linked via an azo, an ester, an anhydride, a thioester or an amide linkage to a molecule of 4- or 5-aminosalicylic acid. Furthermore, the present invention provides a process for preparing these compounds and their use for treating IBD and IBS and the prevention/treatment of colon cancer.

This application is a divisional of U.S. patent application Ser. No.12/368,846, filed Oct. 2, 2009, which is a continuation of U.S. patentapplication Ser. No. 11/278,395, filed Mar. 31, 2006, which is acontinuation-in-part of U.S. patent application Ser. No. 11/139,871,filed May 27, 2005.

FIELD OF THE INVENTION

The present invention relates to compounds useful in the treatment of anintestinal disease such as inflammatory bowel disease (IBD) andirritable bowel syndrome (IBS) and in colon cancer chemoprevention. Inparticular, 4- and 5-aminosalicylic acid derivatives have been developedhaving a hydrogen sulfide releasing moiety linked via an azo, an ester,an anhydride, a thioester or an amide linkage to a molecule of 5- or4-aminosalicylic acid.

BACKGROUND OF THE INVENTION

Inflammatory bowel disease (IBD) is the general name for diseases thatcause inflammation in the small intestine and colon. Ulcerative colitisis the most common inflammatory bowel disease and it affects variousportions of the gastrointestinal (GI) tract, particularly the lower GItract, and more particularly the colon and/or rectum. A second IBD isCrohn's disease, which predominates in the small intestine (ileum) andthe large intestine (colon).

Ulcerative colitis can be difficult to diagnose in that its symptoms aresimilar to other intestinal disorders and to Crohn's disease. Crohn'sdisease differs from ulcerative colitis because it causes deeperinflammation into the intestinal wall. Also, Crohn's disease usuallyoccurs in the small intestine, although it can also occur in the mouth,esophagus, stomach, duodenum, large intestine, appendix, and anus.

Ulcerative colitis may occur in people of any age, but most often itstarts between ages 15 and 30, or less frequently between ages 50 and70. Children and adolescents sometimes develop this disease. Ulcerativecolitis affects men and women equally and appears to run in somefamilies.

It is also important to consider that about 5 percent of people withulcerative colitis develop colon cancer. The risk of cancer increaseswith the duration and the extent of involvement of the colon. Forexample, if only the lower colon and rectum are involved, the risk ofcancer is no higher than normal. However, if the entire colon isinvolved, the risk of cancer may be as much as 32 times the normal rate.Thus, it is possible that drugs useful in the treatment of IBD may alsobe useful in the prevention of colon cancer.

The pathogenesis of IBD likely involves multifactorial interactionsamong genetic factors, immunological factors and environmental triggers.Recent evidence suggests that a pathologic activation of the mucosalimmune system in response to antigens is a key factor in thepathogenesis of IBD.

The presentation of antigen in the inflammatory process is closelyfollowed by generation of cytokines, small glycoprotein peptidemolecules, which provide signals for the communication among differentcell populations determining the direction of subsequent immune andinflammatory response. Pro-inflammatory cytokines include interleukin(IL)-1, IL-6, IL-8 and tumor necrosis factor-alpha (TNF-α). Macrophagesare the major source of cytokines, with epithelial cells also being ableto produce a number of these peptide factors.

T helper (Th) cells are a further important source of cytokines. Th1cells, which are associated with a cell-mediated immune response,produce IL-2, interferon gamma (IFN-□γ) and TNF-α. A key transcriptionfactor involved in the regulation of inflammation, NFkB, which isspecifically implicated in the pathogenesis of IBD, regulates the amountof cytokines produced by the Th1 cells (see Neurath et al. (1996) NatureMed. 2: 998-1004). Th2 cells enhance antibody synthesis by B cells andproduce IL-4, IL-5, IL-6, and IL-10.

Chemokines are also thought to contribute to the pathogenesis ofcolitis. Chemokines are pro-inflammatory proteins that participate inimmune and inflammatory responses through the chemoattraction andactivation of leukocytes. For example, RANTES is a C—C chemokine thatpromotes the recruitment and activation of inflammatory cells such asmonocytes, lymphocytes, mast cells and eosinophils. RANTES has recentlybeen shown to be elevated during the chronic phase of colitis (seeAjuebor et al. (2001) J. Immunol. 166: 552-558).

Treatment for ulcerative colitis depends on the seriousness of theillness. Most people are treated with medication. In severe cases, apatient may need surgery to remove the diseased colon.

Irritable bowel syndrome (IBS) is a common but poorly understooddisorder that causes a variety of bowel symptoms including abdominalpain, diarrhea and/or constipation, bloating, gassiness and cramping.While these symptoms may be caused by a number of different boweldiseases, IBS is usually diagnosed only after exclusion of a moreserious problem. There is increasing evidence suggesting the role ofinflammation in the pathogenesis of IBS.

The goal of therapy is to induce and maintain remission, and to improvethe quality of life for people with IBD/IBS. Several types of drugs areavailable.

Aminosalicylates, which are drugs that contain 5-aminosalicylic acid(5-ASA; mesalamine) or 4-aminosalicylic acid (4-ASA), help to controlthe inflammation. However, both mesalamine and 4-ASA may be absorbed asit passes through the GI tract and may adversely affect the amount ofmesalamine that reaches the lower GI tract, particularly the colon andrectum. Thus, various mesalamine formulations have been introduced in anattempt to protect mesalamine as it passes through the gut and upper GItract.

In addition, several pro-drugs of mesalamine have been introduced whichcan aid in colon-specific delivery of mesalamine. These pro-drugs aregenerally less readily absorbed in the gut and upper GI tract and thuscan more easily reach the colon.

Sulfasalazine is a combination of sulfapyridine and 5-ASA and isemployed to induce and maintain remission. Sulfasalazine is metabolizedin the body to form 5-ASA and sulfapyridine. The sulfapyridine componentcarries the anti-inflammatory 5-ASA to the intestine.

However, sulfapyridine may lead to side effects, such as nausea,vomiting, heartburn, diarrhea, and headache. These adverse side effectsare usually attributed to the activity of sulfapyridine in the GI tract,as well as that absorbed into the system.

Other 5-ASA agents such as olsalazine, ipsalazide and balsalazide, eachof which have a different carrier, offer fewer side effects, and may beused by people who cannot take sulfasalazine. Unlike sulfasalazine, thebreakdown of these 5-ASA compounds in the intestinal tract may not giverise to undesirable metabolic products.

In general, 5-ASA compounds are given orally, through an enema, or in asuppository, depending on the location of the inflammation in the colon.Most people with mild or moderate ulcerative colitis are treated withthis group of drugs first. However, in general, this therapy cannot beconsidered optimal, mainly because of the poor potency of the drug thatcauses also a poor compliance for the patient.

Other drugs that are used are corticosteroids such as prednisone,hydrocortisone, budesonide etc. and immunomodulators such asazathioprine and 6-mercaptopurine (6-MP). These drugs can cause sideeffects such as hypertension, increased risk of infections etc.

Sulfasalazine, olsalazide and balsalazide are mesalamine derivativeswhere the non-mesalamine carrier is linked to mesalamine via a diazobond. These pro-drugs are not as readily absorbed in the gut and upperGI tract and thus can reach the colon where they are split byazo-reductases of the colonic microflora to release the mesalamine andcarrier directly in the colon.

Other derivatives of mesalamine comprise a carrier attached tomesalamine via the carboxylic and hydroxyl functional groups of themolecule. Among these, the preparation of esters or amides with aminoacids such as L-serine and L-glycine or the addition of other biologicalcompound such as taurine has been reported. These pro-drugs base theiractivity on the action of carboxypeptidases and aminopeptidases A forreleasing mesalamine. (R. Pellicciari et al. (1993) Journal of MedicinalChemistry, 36, pg. 4201-7).

Most of the prior art carrier moieties attached to mesalamine are inert.Thus, it is desirable to link carrier moieties to either 5-ASA or 4-ASA,which are also biologically active and useful in the treatment ofIBD/IBS.

SUMMARY OF THE INVENTION

In general, a hydrogen sulfide (H₂S) releasing moiety that is capable ofreleasing H₂S in tissue is linked via an azo, an ester, an anhydride, athioester or an amide linkage to a molecule of 4- or 5-aminosalicylicacid (4- or 5-ASA) to form a 4- or 5-ASA derivative of the presentinvention. By covalently attaching the H₂S releasing moiety to 4- or5-ASA, the derivatives of the present invention may act as pro-drugsthat are generally less readily absorbed in the gut and upper GI tractand thus can more easily reach the colon.

The anti-inflammatory properties of 4- or 5-ASA and their use to treatulcerative colitis are well documented. 4- or 5-ASA reduces bowelinflammation, diahhrea (stool frequency), rectal bleeding and stomachpain. H₂S has been recently shown to function as a neuromodulatory andto exert anti-inflammatory actions. Further, H₂S has been shown tomodulate nociception to colorectal distention (see Distrutti et al.(2005) Evidence That Hydrogen Sulfide Exerts Antinociceptive Effects inthe Gastrointestinal Tract by Activating K_(ATP) Channels. J. Pharm. andExp. Ther. 316: 325-335, incorporated herein by reference). Finally, H₂Shas been shown to be a smooth muscle relaxant in intestinal tissues (seeTeague, B. et al. (2002) The Smooth Muscle Relaxant effect of HydrogenSulfide In Vitro: Evidence for a Physiological Role to ControlIntestinal Contractility. Br. J. Pharmacol. 137: 139-145, incorporatedherein by reference).

Surprisingly, covalently attaching the H₂S releasing moiety to 4- or5-ASA improves the H₂S releasing capabilities of the H₂S moiety ascompared to the H₂S moiety alone. This suggests that H₂S may be releasedboth while the H₂S releasing moiety is covalently attached to 4- or5-ASA as well as after the H₂S releasing moiety is cleaved from the 4-or 5-ASA by hydrolysis or cleavage by various enzymes present in the GItract to release the two active ingredients, namely, 4- or5-aminosalicylic acid and the H₂S releasing moiety for further action.

The derivatives of the present invention are superior to 4- or 5-ASAalone, H₂S releasing moiety alone and a mixture of 4- or 5-ASA and H₂Sreleasing moiety in reducing inflammation, reducing diahhrea and fecaloccult blood in subjects with colitis, and reducing visceral painassociated with colorectal distention. Further, the derivatives of thepresent invention also reduce mRNA levels of cyclooxygenase (COX)-1,COX-2, constitutive endothelial nitric oxide synthase (eNOS), andinducible NOS (iNOS), all of which are enzymes believed to be involvedin inflammation.

Thus, in one aspect of the invention, the derivatives of the presentinvention are useful in treating an inflammatory condition of thegastrointestinal (GI) tract, such as inflammatory bowel disease (IBD)and irritable bowel syndrome (IBS). Without being bound to theory, it isthought that the hydrogen sulfide released from the hydrogen sulfidereleasing moiety exerts anti-inflammatory effects via the inhibition ofNFkB, the transcription factor that regulates the expression of severalof the pro-inflammatory genes. Further, it is thought that theantinociceptive effects of H₂S may involve the ATP-sensitive K⁺(K_(ATP)) channels.

In another aspect of the invention, the 4- or 5-ASA derivatives of thepresent invention are effective in decreasing the viability of HT-29human colon cancer cells and thus are useful in the prevention and/ortreatment of colon cancer.

Broadly stated, compounds of the invention have the following generalformula:

A-L-R  (I)

where:

A is

-   -   where —N═ is either at position 4 or 5,

-   -   where —NH is either at position 4 or 5,

-   -   where —NH₂ is either at position 4 or 5,        or

-   -   where —NH₂ is either at position 4 or 5;        L is either O, O—C═O, S, N or a covalent bond to form an ester        linkage, an anhydride linkage, a thioester linkage, an amide        linkage or an azo linkage; and        R is a hydrogen sulfide releasing moiety that releases H₂S in        tissue. It is understood that any non-toxic, effective hydrogen        sulfide releasing moiety that releases H2S in the presence of        tissue can be used in the present invention.

In a preferred embodiment, R is selected from the group consisting of:

All of the above moieties release H₂S in biological tissues; however,most of the H₂S releasing moieties do so by a different mechanism thanN-acetylcysteine. It is well known that N-acetylcysteine is converted tocysteine in various tissues, and that cysteine metabolism in vivoproduces H₂S. H₂S is produced mainly by two types of pyridoxal5′-phosphate dependent enzymes responsible for metabolism of L-cysteine,namely, cystathione γ-lyase and cystathione β-synthase (see Fujii et al.(2005) Hydrogen Sulfide as an Endogenous Modulator of BiliaryBicarbonate Excretion in the Rat Liver. Antioxid. Redox Signal. 7:788-794, incorporated herein by reference).

Pharmaceutical acceptable salts such as for example salts with alkalinemetals and alkaline earth metals, non-toxic amines and amino acids arealso part of the present invention. Preferred salts are the salts witharginine and agmatine. Also included are pharmaceutically acceptableacid addition salts.

In a further aspect the present invention provides a pharmaceuticalcomposition of the compounds of the present invention, and apharmaceutically acceptable excipient or carrier, particularly one foruse in the treatment of an inflammatory condition of the GI tract.

According to other embodiments of the present invention, methods oftreating an inflammatory condition of the GI tract, such as inflammatorybowel disease (IBD) and irritable bowel syndrome (IBS), in a subject inneed of such treatment, include administering to the subject aneffective amount of 4- or 5-ASA derivatives and their salts. Further,methods for the treatment or prevention of colon cancer in a subject inneed thereof are provided comprising administering to the subject aneffective amount of 4- or 5-ASA derivatives and their salts.

In a further embodiment, the present invention provides the use of 4- or5-ASA derivatives and their salts of the present invention for themanufacture of a medicament for the treatment of an inflammatorycondition of the GI tract. The present invention also provides the useof 4- or 5-ASA derivatives and their salts for the treatment of aninflammatory condition of the GI tract.

Preferred compounds are those of the following formulae:

2-Hydroxy-4- or 5-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenylazo]-benzoicacid (II)

4- or 5-Amino-2-hydroxy-benzoic acid4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester (III)

4 or5-Amino-2-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenoxycarbonyloxy]-benzoicacid (IV)

2-Hydroxy-4 or5[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenoxycarbonylamino]-benzoic acid(V)

4- or5-{[(1-Carboxy-2-mercapto-ethylcarbamoyl)-methyl]-azo}-2-hydroxy-benzoicacid (VI)

4- or 5-Amino-2-hydroxy-benzoic acid(1-carboxy-2-mercapto-ethylcarbamoyl)-methyl ester (VII)

4- or5-Amino-2-[(1-carboxy-2-mercapto-ethylcarbamoyl)-methoxycarbonyloxy]-benzoicacid (VIII)

4- or5-[(1-Carboxy-2-mercapto-ethylcarbamoyl)-methoxycarbonylamino]-2-hydroxy-benzoicacid (IX)

4- or 5-amino-2-hydroxy-benzoic acid anhydride with N-acetylcysteine(X),

4- or 5-(2-Acetylamino-3-mercapto-propionylamino)-2-hydroxy-benzoic acid(XI)

2-(2-Acetylamino-3-mercapto-propionyloxy)-4 or 5-amino-benzoic acid(XII)

2-Hydroxy-4 or5-({4-[4-(4-methoxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenoxymethyl}-azo)-benzoicacid (XIII)

4- or5-Amino-2-{4-[4-(4-methoxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenoxymethoxycarbonyloxy}-benzoicacid (XIV)

2-Hydroxy-4- or5-{4-[4-(4-methoxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenoxymethoxycarbonylamino}-benzoicacid (XV)

4- or 5-Amino-2-hydroxy-benzoic acid4-[4-(4-methoxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenoxymethylester (XVI)

4- or 5-Amino-2-hydroxy-benzoic acid4-[4-(4-hydroxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenylester (XVII)

4- or5-Amino-2-{4-[4-(4-hydroxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenoxycarbonyloxy}-benzoicacid (XVIII)

2-Hydroxy-4- or5-{4-[4-(4-hydroxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenoxycarbonylamino}-benzoicacid (XIX)

4- or 5-(1-Carboxy-3-thiocarbamoyl-propylazo)-2-hydroxy-benzoic acid(XX)

2-(4- or 5-Amino-2-hydroxy-benzoylamino)-4-thiocarbamoyl-butyric acid(XXI)

4- or 5-Amino-2-(1-carboxy-3-thiocarbamoyl-propylcarbamoyloxy)-benzoicacid (XXII)

2-Hydroxy-4- or5-[3-(1-hydroxymethyl-3-thiocarbamoyl-propyl)-ureido]-benzoic acid(XXIII)

4- or 5-Amino-2-(2-amino-4-thiocarbamoyl-butyryloxy)-benzoic acid (XXIV)

4- or 5-(2-Amino-4-thiocarbamoyl-butyrylamino)-2-hydroxy-benzoic acid(XXV)

4- or 5-Amino-2-hydroxy-benzoic acid anhydride with2-amino-4-thiocarbamoyl-butyric acid (XXVI)

4-thiocarbamoylphenyl 4- or 5-amino-2-hydroxybenzoate (XXVII)

4- or 5-Amino-2-(4-thiocarbamoyl-phenoxycarbonyloxy)-benzoic acid(XXVIII)

2-Hydroxy-4- or 5-(4-thiocarbamoyl-phenoxycarbonylamino)-benzoic acid(XXIX)

4- or 5-Amino-2-hydroxy-benzoic acid thiocarbamoylmethyl ester (XXX)

4- or 5-Amino-2-thiocarbamoylmethoxycarbonyloxy-benzoic acid (XXXI)

2-Hydroxy-4- or 5-thiocarbamoylmethoxycarbonylamino-benzoic acid (XXXII)

4- or 5-Amino-2-hydroxy-benzoic acid anhydride with sulfuric acidmono-(2-mercapto-ethyl)ester (XXXIII)

4- or 5-Amino-2-(2-mercapto-ethoxysulfonyloxy)-benzoic acid (XXXIV), and

5-Amino-2-hydroxy-benzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenylester (XXXV)

The most preferred compounds are as follows:

5-Amino-2-hydroxy-benzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenylester (XXXV)

4-thiocarbamoylphenyl 4- or 5-amino-2-hydroxybenzoate (XXVII); and

4- or 5-amino-2-hydroxy-benzoic acid anhydride with N-acetylcysteine (X)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the Disease Activity Score of mice having TNBS-inducedcolitis after treatment with increasing doses of mesalamine and CompoundXXXV of the present invention.

FIG. 2 shows the myeloperoxidase (MPO) activity in mice havingTNBS-induced colitis after treatment with increasing doses of mesalamineand Compound XXXV of the present invention.

FIG. 3 shows the Disease Activity Score of mice having TNBS-inducedcolitis after treatment with Compound XXXV, mesalamine alone,5-p-hydroxyphenyl-1,2-dithione-3-thione (ADT-OH) alone and a mixture ofmesalamine and ADT-OH.

FIG. 4 shows the myeloperoxidase (MPO) activity in mice havingTNBS-induced colitis after treatment with Compound XXXV, mesalaminealone, ADT-OH alone and a mixture of mesalamine and ADT-OH.

FIG. 5 shows the Disease Activity Score of mice having TNBS-inducedcolitis after treatment with Compound XXVII, mesalamine alone,4-hydroxythiobenzamide (4-HTB) alone and a mixture of mesalamine and4-HTB.

FIG. 6 shows the myeloperoxidase (MPO) activity in mice havingTNBS-induced colitis after treatment with Compound XXVII, mesalaminealone, 4-HTB alone and a mixture of mesalamine and 4-HTB.

FIG. 7 shows the myeloperoxidase (MPO) activity in mice havingTNBS-induced colitis after treatment with 50 mg/kg mesalamine andequimolar dose of Compound XXXV of the present invention.

FIG. 8 shows the Disease Activity Score of mice having TNBS-inducedcolitis after treatment with vehicle (1% CMC), 50 mg/kg mesalamine andequimolar dose of Compound XXXV of the present invention.

FIG. 9 shows colonic tumour necrosis factor (TNF-α) mRNA expression inmice with TNBS-induced colitis after treatment with vehicle (1% CMC), 50mg/kg mesalamine and equimolar dose of Compound XXXV of the presentinvention.

FIG. 10 shows interferon gamma (IFN-γ) mRNA expression in mice withTNBS-induced colitis after treatment with vehicle (1% CMC), 50 mg/kgmesalamine and equimolar dose of Compound XXXV of the present invention.

FIG. 11 shows various interleukin (IL) mRNA expression, namely, IL-1,-2, 10 and -12 mRNA, in mice with TNBS-induced colitis after treatmentwith vehicle (1% CMC), 50 mg/kg mesalamine and equimolar dose ofCompound XXXV of the present invention.

FIG. 12 shows colonic levels of RANTES mRNA in mice with TNBS-inducedcolitis after treatment with vehicle (1% CMC), 50 mg/kg mesalamine andequimolar dose of Compound XXXV of the present invention.

FIG. 13 shows colonic COX-1 and COX-2 mRNA expression in mice withTNBS-induced colitis after treatment with vehicle (1% CMC), 50 mg/kgmesalamine and equimolar dose of Compound XXXV of the present invention.

FIG. 14 shows colonic eNOS and iNOS mRNA expression in mice withTNBS-induced colitis after treatment with vehicle (1% CMC), 50 mg/kgmesalamine and equimolar dose of Compound XXXV of the present invention.

FIGS. 15( a) and (b) show the perception score in a rat model ofvisceral pain perception using mesalamine and Compound XXXV,respectively, of the present invention.

FIGS. 16( a) and (b) show the intrarectal pressure in a rat model ofvisceral pain perception using mesalamine and Compound XXXV,respectively, of the present invention.

FIG. 17 shows the pain perception scores of mesalamine, Compound XXXVand Compound XXVII with or without glibenclamide.

FIG. 18 shows the pain perception scores of Compound XXXV, CompoundXXVII, mesalamine, ADT-OH and 4-HBT.

FIG. 19 shows leukocyte adherence in response to intragastricadministration of aspirin.

FIG. 20 is a bar graph of the leukocyte adherence for the final timeperiod of the experiment (minutes 60-65).

FIG. 21 is a bar graph showing H2S generation of cysteine, ADT-OH,Compound XXXV, 4-HTB and Compound XXVII.

FIG. 22 is a Concentration-Response curve showing the vasorelaxanteffects of H₂S releasing moieties of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention will now be described with respect to preferredembodiments described herein. It should be appreciated however thatthese embodiments are for the purpose of illustrating the invention, andare not to be construed as limiting the scope of the invention asdefined by the claims.

The compounds of the present invention contain two active moieties,either 4- or 5-ASA and a hydrogen sulfide releasing moiety, linkedtogether by an azo, ester, anhydride, thioester or amide linkage. Thepresence of azo-reductase enzymes allow for the release of 4- or 5-ASAfrom the azo bond pro-drugs thus allowing a targeted delivery to thecolon and reducing at the same time the systemic absorption. Similarly,the presence of carboxypeptidases and aminopeptidases A also allow forthe release of 4- or 5-ASA from the ester and amide bond pro-drugs,respectively. Esterases and thioesterases will also cleave ester andthioester linkages, respectively. Finally, lipases will cleave anhydridelinkages. The compounds of the present invention can be made using knownstarting materials and reagents.

Compounds of the present invention may be utilized for the prophylaxisor treatment of various diseases, particularly inflammatory conditionsof the GI tract including, but not limited to, inflammatory conditionsof the mouth such as mucositis, infectious diseases (e.g., viral,bacterial and fungal diseases), and Crohn's disease; inflammatoryconditions of the esophagus such as esophagitis, conditions resultingfrom chemical injury (e.g., lye ingestion), gastroesophageal refluxdisease, bile acid reflux, Barrett's esophagus, Crohn's disease, andesophageal stricture; inflammatory conditions such as gastritis (e.g.,Helicobacter pylori, acid-peptic disease and atrophic gastritis), celiacdisease, peptic ulcer disease, pre-cancerous lesions of the stomach,non-ulcer dyspepsia, and Crohn's disease; inflammatory conditions of thestomach such as Crohn's disease, bacterial overgrowth, peptic ulcerdisease, and fissures of the intestine; inflammatory conditions of thecolon such as Crohn's disease, ulcerative colitis, irritable bowelsyndrome, infectious colitis (e.g., pseudomembranous colitis such asClostridium difficile colitis, salmonella enteritis, shigellainfections, yersiniosis, cryptospiridiosis, microspridial infections,and viral infections), radiation-induced colitis, colitis in theimmunocompromised host (e.g., typhlitis), precancerous conditions of thecolon (e.g., dysplasia, inflammatory conditions of the bowel, andcolonic polyps), proctitis, inflammation associated with hemorrhoids,proctalgia fugax, and rectal fissures; liver gallbladder and/or bilarytract conditions such as cholangitis, sclerosing cholangitis, primarybilary cirrhosis, and cholecystitis; and intestinal abscess.

Depending on the specific condition or disease state to be treated,subjects may be administered compounds of the present invention at anysuitable therapeutically effective and safe dosage, as may be readilydetermined within the skill of the art. These compounds are, mostdesirably, administered in dosages ranging from about 1 to about 2000 mgper day, in a single or divided doses, although variations willnecessarily occur depending upon the weight and condition of the subjectbeing treated and the particular route of administration chosen.However, a dosage level that is in the range of about 0.1 to about 100mg/kg, preferably between about 5 and 90 mg/kg, and more preferablybetween about 5 and 50 mg/kg, is most desirable. Variations maynevertheless occur depending upon the weight and conditions of thepersons being treated and their individual responses to said medicament,as well as on the type of pharmaceutical formulation chosen and the timeperiod and interval during which such administration is carried out. Insome instances, dosage levels below the lower limit of the aforesaidrange may be more than adequate, while in other cases still larger dosesmay be employed without causing any harmful side effects, provided thatsuch large doses are first divided into several small doses foradministration throughout the day.

The compounds of the present invention can be administered in the formof any pharmaceutical formulation, the nature of which will depend uponthe route of administration. These pharmaceutical compositions can beprepared by conventional methods, using compatible, pharmaceuticallyacceptable excipients or vehicles. Examples of such compositions includecapsules, tablets, transdermal patches, lozenges, troches, sprays,syrups, powders, granulates, gels, elixirs, suppositories, and the like,for the preparation of extemporaneous solutions, injectablepreparations, rectal, nasal, ocular, vaginal etc. A preferred route ofadministration is the oral and rectal route.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (preferably corn, potato or tapioca starch), alginic acidand certain complex silicates, together with granulation binders likepolyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc can be used for tabletting purposes. Solid compositions of similartype may also be employed as fillers in gelatin capsules; preferredmaterials in this connection also include lactose or milk sugar, as wellas high molecular weight polyethylene glycols. When aqueous suspensionsand/or elixirs are desired for oral administration the active ingredientmay be combined with sweetening or flavoring agents, coloring matterand, if so desired, emulsifying and/or suspending agents, together withsuch diluents as water, ethanol, propylene glycol, glycerin and variouscombinations thereof.

The dosage form can be designed for immediate release, controlledrelease, extended release, delayed release or targeted delayed release.The definitions of these terms are known to those skilled in the art.Furthermore, the dosage form release profile can be effected by apolymeric mixture composition, a coated matrix composition, amultiparticulate composition, a coated multiparticulate composition, anion-exchange resin-based composition, an osmosis-based composition, or abiodegradable polymeric composition. Without wishing to be bound bytheory, it is believed that the release may be effected throughfavorable diffusion, dissolution, erosion, ion-exchange, osmosis orcombinations thereof.

For parenteral administration, a solution of an active compound ineither sesame or peanut oil or in aqueous propylene glycol can beemployed. The aqueous solutions should be suitably buffered (preferablypH greater than 8), if necessary, and the liquid diluent first renderedisotonic. The aqueous solutions are suitable for intravenous injectionpurposes. The preparation of all these solutions under sterileconditions is readily accomplished by standard pharmaceutical techniqueswell known to those skilled in the art.

The following non-limitative examples further describe and enable aperson ordinarily skilled in the art to make and use the invention.

Preparation of Compounds Example 1 Synthesis of2-Hydroxy-5-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenylazo]-benzoic acid(4) [Compound of Formula II]

Synthesis of (4-Propenyl-phenyl)-carbamic acid tert-butyl ester (2)

To the solution of 4-propenyl-phenylamine (1) (10.0 mmol) in 25 mL ofdioxane and 12.5 mL of water, triethylamine (15.0 mmol) anddi-tert-butyl-dicarbonate (15.0 mmol) were added with stirring at 0° C.for ½ h. The reaction mixture was stirred mechanically for 24 h at roomtemperature. After evaporation of the solvent, 3 M HCl (15 mL), wasadded drop wise to the residue. The precipitate is filtered, washed withwater and dried. The residue was loaded on a silica gel open column andeluted with CH₂Cl₂/MeOH (9/1), from which (4-Propenyl-phenyl)-carbamicacid tert-butyl ester (2) was obtained (90% yield).

Synthesis of 5-(4-Amino-phenyl)-[1,2]dithiole-3-thione (3)

(4-Propenyl-phenyl)-carbamic acid tert-butyl ester (2, 4.5 mmol) andsulphur (31.5 mmol) were heated in dimethyl formamide (500 ml) for 8 hr;the residue after removal of solvent was almost completely soluble intoluene. An attempt to extract the toluene liquors with 2N aqueoussodium hydroxide, gave a precipitate of an orange solid. This productwas dissolved in boiling water, treated with 4N hydrochloric acid for 30min at room temperature; addition of 4N NaOH furnished the desiredproduct (3) (yield 55%).

Synthesis of2-Hydroxy-5-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenylazo]-benzoic acid(4)

5-(4-Amino-phenyl)-[1,2]dithiole-3-thione (3, 0.56 mmol) was dissolvedin a mixture of 5 mL of concentrated HCl and 2.5 mL of water anddiazotized with a solution of sodium nitrite (0.56 mmol). In themeantime salicylic acid (0.56 mmol), potassium hydroxide (1.12 mmol) andsodium carbonate are dissolved in water. The diazo suspension is addedin portions to the alkaline solution of salicylic acid and thealkalinity maintained at a sufficiently high level during the wholereaction by means of addition of further quantities of potassiumhydroxide solution. After 2 days the reaction mixture is heated for 30min at 50° C. The azo compound (4) was precipitated by means of HCl andfiltered off (yield 85%), to yield the compound of Formula II,2-Hydroxy-5-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenylazo]-benzoic acid.

Example 2 Synthesis of2-Hydroxy-4-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenylazo]-benzoic acid(2) [Compound of Formula II]

Synthesis of2-Hydroxy-4-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenylazo]-benzoic acid(2)

4-Amino-2-hydroxy-benzoic acid (1, 1 mmol) was dissolved in a mixture of10 mL of concentrated HCl and 5 mL of water and diazotized with asolution of sodium nitrite (1 mmol). The diazo suspension is added inportions to a solution of 5-phenyl-[1,2]dithiole-3-thione (1 mmol) indimethylformamide. After 2 days the reaction mixture is heated for 30min at 50° C. After cooling the azo compound (2) was precipitated bymeans of HCl and filtered off (yield 65%), to yield the compound ofFormula II,2-Hydroxy-5-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenylazo]-benzoic acid.

Example 3 General Synthetic Procedure of 4- or 5-Amino-2-hydroxy-benzoicacid 4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester (4) [Compound ofFormula XXXV]

Synthesis of 5-p-hydroxyphenyl-1,2-dithione-3-thione (ADT-OH)

Anethole (1) (32.5 g; 0.21 mol) and sulphur (45 g; 1.40 mol) were heatedin dimethylformamide (250 ml) for 8 hr; the residue after removal ofsolvent was almost completely soluble in toluene. An attempt to extractthe toluene liquors with 2N-aqueous sodium hydroxide, gave a precipitateof an orange solid (8.5 g). m.p. over 300° C. This product was dissolvedin boiling water and gave an orange precipitate (2) after addition ofhydrochloric acid (Yield 50%), m.p. 188-189° C. ¹H NMR (DMSO) δ 6.86 (d,2H), 7.68 (s, 1H), 7.75 (d, 2H), 10.51 (s, —OH); MS (ESI), m/z 225 (M⁻).

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1)

To the solution of 4- or 5-amino salicylic acid (10.0 mmol) in 25 mL ofdioxane and 12.5 mL of water, triethylamine (15.0 mmol) anddi-tert-butyl-dicarbonate (15.0 mmol) were added with stirring at 0° C.for ½ h. The reaction mixture was stirred mechanically for 24 h at roomtemperature. After evaporation of the solvent, 3M HCl (15 mL), was addeddrop wise to the residue. The precipitate is filtered, washed with waterand dried. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (9/1), from which 4- or5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1) was obtained (80%yield).

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-tert-butoxy-benzoic acid(2)

Compound (1) (12.0 mmol), conc. H₂SO₄ (6.0 mmol), and DCM (100 mL) werestirred under isobutylene gas (5 psi) for 6 h at room temperature. Thesolution was washed with cold 10% NaHCO₃ (2×100 mL) and brine (100 mL),dried (Na₂SO₄) and evaporated. The residue was dissolved in 1:1MeOH/CCl₄ (400 mL), washed with water (300 mL), and then extracted with1:1 MeOH/water (2×200 mL). The extract was dried (Na₂SO₄) and evaporatedto a white solid (2), which was recrystallized by DCM/hexane (83%yield).

Synthesis of 4- or 5-Amino-2-hydroxy-benzoic acid4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester (4)

To the solution of 4- or 5-tert-butoxycarbonylamino-2-hydroxy-benzoicacid (2) (3.0 mmol) in 50 mL of dimethylformamide, hydroxybenzotriazole(3.3 mmol) and DCC (3.3 mmol) were added with stirring at 0° C. for 1 h.To the reaction mixture, 5-p-hydroxyphenyl-1,2-dithione-3-thione(ADT-OH) (3.0 mmol) was added and stirred mechanically for 3 h at 0° C.and 72 h at room temperature. After filtration, the filtrate wasevaporated under reduced pressure to remove the solvent. The oilyresidue thus obtained was dissolved in ethyl acetate; the organic layerwas washed with brine, dried on anhydrous MgSO₄, filtered and thesolvent evaporated. The crude intermediate (3) was treated with asolution of 40% TFA in CH₂Cl₂. After 2 h the solvent was removed toobtain compound 3 as a crude residue. The residue was loaded on a silicagel open column and eluted with CH₂Cl₂/MeOH (8/2), from which 4- or5-amino-2-hydroxy-benzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenylester (4) [compound of Formula XXXV] was obtained (40% yield).

Compound 5-amino-2-hydroxy-benzoic acid4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester (4): ¹H NMR (DMSO) δ 7.07(d, 2H), 7.38 (d, 2H), 7.46 (d, 2H), 7.79 (s, 1H), 7.85 (s, 1H), 8.01(d, 2H), 10.35 (s, —OH); MS (ESI), m/z 362 (M⁺).

Example 4 General Synthetic Procedure of 2-(tert-butoxycarbonyl)-4- or5-aminophenyl hydrogen carbonate (5) 3-(tert-butoxycarbonyl)-4- or5-hydroxyphenyl-carbamic acid (6)

Synthesis of 4- or 5-Amino-2-ethoxycarbonyloxy-benzoic acid (1) and 4-or 5-ethoxycarbonylamino-2-hydroxy-benzoic acid (2)

4- or 5-amino salicylic acid (3.0 mmol) was dissolved in 40 mLchloroform in round-bottomed flask fitted with a drying tube. Ethylchloroformate (3.0 mmol) was added gradually and the solution refluxedfor 2 hours. The chloroform was evaporated in vacuo and the residue thentaken up in ether. The ether phase was decolorized using charcoal,filtered and the solvent removed in vacuo. The residue obtained was thendissolved in ethanol and the product recovered by precipitation usingn-hexane as a crude oily semisolid. The crude product was purified byFlash-chromatography on silica gel eluting with diethyl ether/hexane(7:3, v/v) to obtain the title compounds: 4- or5-Amino-2-ethoxycarbonyloxy-benzoic acid (1: yield: 58%) and 4- or5-Ethoxycarbonylamino-2-hydroxy-benzoic acid (2: yield: 34%).

Synthesis of 4- or 5-Amino-2-ethoxycarbonyloxy-benzoic acid tert-butylester (3)

To the solution of (1) (3.0 mmol) in 50 mL of dimethylformamide,hydroxybenzotriazole (3.3 mmol) and DCC (3.3 mmol) were added withstirring at 0° C. for 1 h. To the reaction mixture, t-butanol (3.0 mmol)was added and stirred mechanically for 3 h at 0° C. and 72 h at roomtemperature. After filtration, the filtrate was evaporated under reducedpressure to remove the solvent. The oily residue thus obtained wasdissolved in ethyl acetate; the organic layer was washed with brine,dried on anhydrous MgSO₄, filtered and the solvent evaporated. Theresidue was loaded on a silica gel open column and eluted withCH₂Cl₂/MeOH (9.5/0.5), from which 4- or5-Amino-2-ethoxycarbonyloxy-benzoic acid tert-butyl ester (3) wasobtained (55% yield).

Synthesis of 4- or 5-ethoxycarbonylamino-2-hydroxy-benzoic acidtert-butyl ester (4)

Compound (4) was obtained according the procedure reported to obtaincompound (3). Yield: 74%

Synthesis of 2-(tert-butoxycarbonyl)-4- or 5-aminophenyl hydrogencarbonate (5)

To a solution of the compound (3) (3.5 g; 0.011 mol) in ethanol (80 ml)was added NaOH 1N (40 ml). The reaction mixture was stirred 2 h at roomtemperature. Then the solution was made neutral with HCl 1N. Ethanol wasremoved and the extraction performed with ethyl acetate (3×150 ml); theorganic layers were washed with brine, dried on anhydrous MgSO₄,filtered and the solvent evaporated: 2-(tert-butoxycarbonyl)-4- or5-aminophenyl hydrogen carbonate (5) was obtained (3 g; 0.010 mol;yield: 89%) as a white solid.

Synthesis of 3-(tert-butoxycarbonyl)-4- or 5-hydroxyphenyl-carbamic acid(6)

Compound (6) was obtained according the procedure reported to obtaincompound (5). Yield: 91%

Example 5 General Synthetic Procedure of 4- or5-Amino-2-(1-carboxy-3-thiocarbamoyl-propylcarbamoyloxy)-benzoic acid(11) [Compound of Formula XXII] 4- or5-[3-(1-Carboxy-3-thiocarbamoyl-propyl)-ureido]-2-hydroxy-benzoic acid(12)

Synthesis of 5-thio-L-glutamine-OtBu (2)

L-glutamine-OtBu.HCl (1) (1.2 mmol; 0.3 g) and Lawesson's reagent (0.75mmol; 0.3 g) were added to benzene (20 mL) and the mixture was heatedunder reflux for 15 min. The reaction was then cooled and evaporatedunder vacuum. The crude product was chromatographed over 100 g of silicagel eluted with mixtures of ethyl acetate and n-hexane. There wasobtained 0.2 g (76% yield) of product (2) as a white solid: ¹H NMR(CDCl₃) δ 1.4 (s, 9H), 1.8-2.8 (m, 5H), 4.0-4.8 (m, 3H); MS (ESI), m/z219 (M⁺)

Synthesis of 2-(tert-butoxycarbonyl)-4- or5-tert-Butoxycarbonylaminophenyl hydrogen carbonate (7)

To the solution of (5) (10.0 mmol) in 25 mL of dioxane and 12.5 mL ofwater, triethylamine (15.0 mmol) and di-tert-butyl-dicarbonate (15.0mmol) were added with stirring at 0° C. for ½ h. The reaction mixturewas stirred mechanically for 24 h at room temperature. After evaporationof the solvent, 3 M HCl (15 mL) was added drop wise to the residue. Theprecipitate is filtered, washed with water and dried. The residue wasloaded on a silica gel open column and eluted with CH₂Cl₂/MeOH (9/1),from which 2-(tert-butoxycarbonyl)-4- or5-tert-Butoxycarbonylaminophenyl hydrogen carbonate (7) was obtained(80% yield).

Synthesis of 3-(tert-butoxycarbonyl)-4- or 5-hydroxyphenyl-carbamic acid(8)

Compound 6 (12.0 mmol), conc. H₂SO₄ (6.0 mmol), and DCM (100 mL) werestirred under isobutylene gas (5 psi) for 6 h at room temperature. Thesolution was washed with cold 10% NaHCO₃ (2×100 mL) and brine (100 mL),dried (Na₂SO₄) and evaporated. The residue was dissolved in 1:1MeOH/CCl₄ (400 mL), washed with water (300 mL), and then extracted with1:1 MeOH/water (2×200 mL). The extract was dried (Na₂SO₄) and evaporatedto a white solid (8), which was recrystallized by DCM/hexane (83%yield).

Synthesis of 4- or5-Amino-2-(1-carboxy-3-thiocarbamoyl-propylcarbamoyloxy)-benzoic acid(11)

To the solution of (7) (3.0 mmol) in 50 mL of dimethylformamide,hydroxybenzotriazole (3.3 mmol) and DCC (3.3 mmol) were added withstirring at 0° C. for 1 h. To the reaction mixture,2-amino-4-thiocarbamoyl-butyric acid tert-butyl ester (3.0 mmol) andtriethylamine (3.0 mmol) were added and stirred mechanically for 3 h at0° C. and 72 h at room temperature. After filtration, the filtrate wasevaporated under reduced pressure to remove the solvent. The oilyresidue thus obtained was dissolved in ethyl acetate; the organic layerwas washed with brine, dried on anhydrous MgSO₄, filtered and thesolvent evaporated. The crude intermediate (9) was treated with asolution of 40% TFA in CH₂Cl₂. After 2 h the solvent was removed toobtain compound (11) as a crude residue. The residue was loaded on asilica gel open column and eluted with CH₂Cl₂/MeOH (8/2), from which 4-or 5-Amino-2-(1-carboxy-3-thiocarbamoyl-propylcarbamoyloxy)-benzoic acid(11) was obtained (45% yield), Compound of Formula XXII.

Synthesis of 4- or5-[3-(1-Carboxy-3-thiocarbamoyl-propyl)-ureido]-2-hydroxy-benzoic acid(12)

Compound (12) was obtained according the procedure reported to obtaincompound (11). Yield: 38%

Example 6 General Synthetic Procedure of 4- or5-Amino-2-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenoxycarbonyloxy]-benzoicacid (15) [Compound of Formula IV] 2-Hydroxy-4- or5-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenoxycarbonylamino]-benzoic acid(16) [Compound of Formula V]

Synthesis of 5-p-hydroxyphenyl-1,2-dithione-3-thione (ADT-OH)

Anethole (1) (32.5 g; 0.21 mol) and sulphur (45 g; 1.40 mol) were heatedin dimethylformamide (250 ml) for 8 hr; the residue after removal ofsolvent was almost completely soluble in toluene. An attempt to extractthe toluene liquors with 2 N-aqueous sodium hydroxide, gave aprecipitate of an orange solid (8.5 g). m.p. over 300° C. This productwas dissolved in boiling water and gave an orange precipitate (2) afteraddition of hydrochloric acid (Yield 50%), m.p. 188-189° C. ¹H NMR(DMSO) δ 6.86 (d, 2H), 7.68 (s, 1H), 7.75 (d, 2H), 10.51 (s, —OH); MS(ESI), m/z 225 (M⁻).

Synthesis of 2-(tert-butoxycarbonyl)-4- or5-tert-butoxycarbonylaminophenyl hydrogen carbonate (7)

To the solution of (5) (10.0 mmol) in 25 mL of dioxane and 12.5 mL ofwater, triethylamine (15.0 mmol) and di-tert-butyl-dicarbonate (15.0mmol) were added with stirring at 0° C. for ½ h. The reaction mixturewas stirred mechanically for 24 h at room temperature. After evaporationof the solvent, 3 M HCl (15 mL) was added drop wise to the residue. Theprecipitate is filtered, washed with water and dried. The residue wasloaded on a silica gel open column and eluted with CH₂Cl₂/MeOH (9/1),from which 2-(tert-butoxycarbonyl)-4- or5-tert-Butoxycarbonylaminophenyl hydrogen carbonate (7) was obtained(80% yield).

Synthesis of 3-(tert-butoxycarbonyl)-4- or 5-hydroxyphenyl-carbamic acid(8)

Compound (6) (12.0 mmol), concentrated H₂SO₄ (6.0 mmol), and DCM (100mL) were stirred under isobutene gas (5 psi) for 6 h at roomtemperature. The solution was washed with cold 10% NaHCO₃ (2×100 mL) andbrine (100 mL), dried (Na₂SO₄) and evaporated. The residue was dissolvedin 1:1 MeOH/CCl₄ (400 mL), washed with water (300 mL), and thenextracted with 1:1 MeOH/water (2×200 mL). The extract was dried (Na₂SO₄)and evaporated to a white solid (8), which was recrystallized byDCM/hexane (83% yield).

Synthesis of 4- or5-Amino-2-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenoxycarbonyloxy]-benzoicacid (15)

To the solution of (7) (3.0 mmol) in 50 mL of dimethylformamide,hydroxybenzotriazole (3.3 mmol) and DCC (3.3 mmol) were added withstirring at 0° C. for 1 h. To the reaction mixture,5-p-hydroxyphenyl-1,2-dithione-3-thione (ADT-OH) (3.0 mmol) was addedand stirred mechanically for 3 h at 0° C. and 72 h at room temperature.After filtration, the filtrate was evaporated under reduced pressure toremove the solvent. The oily residue thus obtained was dissolved inethyl acetate; the organic layer was washed with brine, dried onanhydrous MgSO₄, filtered and the solvent evaporated. The crudeintermediate (13) was treated with a solution of 40% TFA in CH₂Cl₂.After 2 h the solvent was removed to obtain compound (15) as a cruderesidue. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (8/2), from which 4- or5-Amino-2-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenoxycarbonyloxy]-benzoicacid (15) was obtained (45% yield), compound of Formula IV.

Synthesis of 2-Hydroxy-4- or5-[4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenoxycarbonylamino]-benzoic acid(16)

Compound (16), compound of Formula V, was obtained according theprocedure reported to obtain compound (15). Yield: 38%

Example 7 General Synthetic Procedure of 4- or5-Amino-2-{4-[4-(4-hydroxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenoxycarbonyloxy}-benzoicacid (19) [Compound of Formula XIV] 2-Hydroxy-4- or5-{4-[4-(4-hydroxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenoxycarbonylamino}-benzoicacid (20) [Compound of Formula XIII]

Synthesis of (p-hydroxyphenyl)dithiophosphonic anhydride

Red P (4 g; 0.129 mol), S (4 g; 0.125 mol) and PhOH (4 g; 0.042 mol)were heated for 5.5 hr at 155-158° C.; the reaction mixture was cooledat room temperature and a precipitate was collected (5.5 g 34% yield).m.p. 224-226° C. The NMR and MS analysis are consistent withp-hydroxyphenyl dithiophosphonic anhydride.

Synthesis of 2-(tert-butoxycarbonyl)-4- or5-tert-butoxycarbonylaminophenyl hydrogen carbonate (7)

To the solution of (5) (10.0 mmol) in 25 mL of dioxane and 12.5 mL ofwater, triethylamine (15.0 mmol) and di-tert-butyl-dicarbonate (15.0mmol) were added with stirring at 0° C. for ½ h. The reaction mixturewas stirred mechanically for 24 h at room temperature. After evaporationof the solvent, 3 M HCl (15 mL) was added drop wise to the residue. Theprecipitate is filtered, washed with water and dried. The residue wasloaded on a silica gel open column and eluted with CH₂Cl₂/MeOH (9/1),from which 2-(tert-butoxycarbonyl)-4- or5-tert-Butoxycarbonylaminophenyl hydrogen carbonate (7) was obtained(80% yield).

Synthesis of 3-(tert-butoxycarbonyl)-4- or 5-hydroxyphenyl-carbamic acid(8)

Compound (6) (12.0 mmol), concentrated H₂SO₄ (6.0 mmol), and DCM (100mL) were stirred under isobutene gas (5 psi) for 6 h at roomtemperature. The solution was washed with cold 10% NaHCO₃ (2×100 mL) andbrine (100 mL), dried (Na₂SO₄) and evaporated. The residue was dissolvedin 1:1 MeOH/CCl₄ (400 mL), washed with water (300 mL), and thenextracted with 1:1 MeOH/water (2×200 mL). The extract was dried (Na₂SO₄)and evaporated to a white solid (8), which was recrystallized byDCM/hexane (83% yield).

Synthesis of 4- or5-Amino-2-{4-[4-(4-hydroxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenoxycarbonyloxy}-benzoicacid (19)

To the solution of (7) (3.0 mmol) in 50 mL of dimethylformamide,hydroxybenzotriazole (3.3 mmol) and DCC (3.3 mmol) were added withstirring at 0° C. for 1 h. To the reaction mixture,p-hydroxyphenyldithiophosphonic anhydride (3.0 mmol) was added andstirred mechanically for 3 h at 0° C. and 72 h at room temperature.After filtration, the filtrate was evaporated under reduced pressure toremove the solvent. The oily residue thus obtained was dissolved inethyl acetate; the organic layer was washed with brine, dried onanhydrous MgSO₄, filtered and the solvent evaporated. The crudeintermediate (17) was treated with a solution of 40% TFA in CH₂Cl₂.After 2 h the solvent was removed to obtain compound (19) as a cruderesidue. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (8/2), from which 4- or5-Amino-2-{4-[4-(4-hydroxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenoxycarboyloxy}-benzoic acid(19) was obtained (65% yield), compound of Formula XIV.

Synthesis of 2-Hydroxy-4- or5-{4-[4-(4-hydroxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenoxycarbonylamino}-benzoicacid (20)

Compound (20), a compound of Formula XIII, was obtained according theprocedure reported to obtain compound (19). Yield: 48%

Example 8 General synthetic procedure of 4- or5-Amino-2-(4-thiocarbamoyl-phenoxycarbonyloxy)-benzoic acid (23)[Compound of Formula XXVIII] 2-Hydroxy-4- or5-(4-thiocarbamoyl-phenoxycarbonylamino)-benzoic acid (24) [Compound ofFormula XXIX]

Synthesis of 2-(tert-butoxycarbonyl)-4- or5-tert-butoxycarbonylaminophenyl hydrogen carbonate (7)

To the solution of (5) (10.0 mmol) in 25 mL of dioxane and 12.5 mL ofwater, triethylamine (15.0 mmol) and di-tert-butyl-dicarbonate (15.0mmol) were added with stirring at 0° C. for ½ h. The reaction mixturewas stirred mechanically for 24 h at room temperature. After evaporationof the solvent, 3 M HCl (15 mL) was added drop wise to the residue. Theprecipitate is filtered, washed with water and dried. The residue wasloaded on a silica gel open column and eluted with CH₂Cl₂/MeOH (9/1),from which 2-(tert-butoxycarbonyl)-4- or5-tert-Butoxycarbonylaminophenyl hydrogen carbonate (7) was obtained(80% yield).

Synthesis of 3-(tert-butoxycarbonyl)-4- or 5-hydroxyphenyl-carbamic acid(8)

Compound (6) (12.0 mmol), concentrated H₂SO₄ (6.0 mmol), and DCM (100mL) were stirred under isobutene gas (5 psi) for 6 h at rt. The solutionwas washed with cold 10% NaHCO₃ (2×100 mL) and brine (100 mL), dried(Na₂SO₄) and evaporated. The residue was dissolved in 1:1 MeOH/CCl₄ (400mL), washed with water (300 mL), and then extracted with 1:1 MeOH/water(2×200 mL). The extract was dried (Na₂SO₄) and evaporated to a whitesolid (8), which was recrystallized by DCM/hexane (83% yield).

Synthesis of 4- or5-Amino-2-(4-thiocarbamoyl-phenoxycarbonyloxy)-benzoic acid (23)

To the solution of (7) (3.0 mmol) in 50 mL of dimethylformamide,hydroxybenzotriazole (3.3 mmol) and DCC (3.3 mmol) were added withstirring at 0° C. for 1 h. To the reaction mixture,4-hydroxy-thiobenzamide (3.0 mmol) was added and stirred mechanicallyfor 3 h at 0° C. and 72 h at room temperature. After filtration, thefiltrate was evaporated under reduced pressure to remove the solvent.The oily residue thus obtained was dissolved in ethyl acetate; theorganic layer was washed with brine, dried on anhydrous MgSO₄, filteredand the solvent evaporated. The crude intermediate (21) was treated witha solution of 40% TFA in CH₂Cl₂. After 2 h the solvent was removed toobtain compound (23) as a crude residue. The residue was loaded on asilica gel open column and eluted with CH₂Cl₂/MeOH (8/2), from which 4-or 5-Amino-2-(4-thiocarbamoyl-phenoxycarbonyloxy)-benzoic acid (23) wasobtained (71% yield), compound of Formula XXVII.

Synthesis of 2-Hydroxy-4- or5-(4-thiocarbamoyl-phenoxycarbonylamino)-benzoic acid (24)

Compound (24), compound of Formula XXIX, was obtained according theprocedure reported to obtain compound (23). Yield: 68%

Example 9 General Synthetic Procedure of 2-(4- or5-Amino-2-hydroxy-benzoylamino)-4-thiocarbamoyl-butyric acid (6)[Compound of Formula XXI]

Synthesis of 5-thio-L-glutamine-OtBu (2)

L-glutamine-OtBu.HCl (1) (1.2 mmol; 0.3 g) and Lawesson's reagent (0.75mmol; 0.3 g) were added to benzene (20 mL) and the mixture was heatedunder reflux for 15 min. The reaction was then cooled and evaporatedunder vacuum. The crude product was chromatographed over 100 g of silicagel eluted with mixtures of ethyl acetate and n-hexane. There wasobtained 0.2 g (76% yield) of product (2) as a white solid: ¹H NMR(CDCl₃) δ 1.4 (s, 9H), 1.8-2.8 (m, 5H), 4.0-4.8 (m, 3H); MS (ESI), m/z219 (M⁺).

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1)

To the solution of 4- or 5-amino salicylic acid (10.0 mmol) in 25 mL ofdioxane and 12.5 mL of water, triethylamine (15.0 mmol) anddi-tert-butyl-dicarbonate (15.0 mmol) were added with stirring at 0° C.for ½ h. The reaction mixture was stirred mechanically for 24 h at roomtemperature. After evaporation of the solvent, 3M HCl (15 mL), was addeddrop wise to the residue. The precipitate is filtered, washed with waterand dried. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (9/1), from which 4- or5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1) was obtained (80%yield).

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-tert-butoxy-benzoic acid(2)

Compound (1) (12.0 mmol), conc. H₂SO₄ (6.0 mmol), and DCM (100 mL) werestirred under isobutylene gas (5 psi) for 6 h at rt. The solution waswashed with cold 10% NaHCO₃ (2×100 mL) and brine (100 mL), dried(Na₂SO₄) and evaporated. The residue was dissolved in 1:1 MeOH/CCl₄ (400mL), washed with water (300 mL), and then extracted with 1:1 MeOH/water(2×200 mL). The extract was dried (Na₂SO₄) and evaporated to a whitesolid (2), which was recrystallized by DCM/hexane (83% yield).

Synthesis of 2-(4- or5-amino-2-hydroxy-benzoylamino)-4-thiocarbamoyl-butyric acid (6)

To the solution of 4- or5-tert-butoxycarbonylamino-2-tert-butoxy-benzoic acid (2) (3.0 mmol) in50 mL of dimethylformamide, hydroxybenzotriazole (3.3 mmol) and DCC (3.3mmol) were added with stirring at 0° C. for 1 h. To the reactionmixture, 2-amino-4-thiocarbamoyl-butyric acid tert-butyl ester (3.0mmol) and triethylamine (3.0 mmol) were added and stirred mechanicallyfor 3 h at 0° C. and 72 h at room temperature. After filtration, thefiltrate was evaporated under reduced pressure to remove the solvent.The oily residue thus obtained was dissolved in ethyl acetate; theorganic layers were washed with brine, dried on anhydrous MgSO₄,filtered and the solvent evaporated. The crude intermediate (5) wastreated with a solution of TFA (40%) in CH₂Cl₂. After 2 h the solventwas removed to obtain compound (6) as a crude residue. The residue wasloaded on a silica gel open column and eluted with CH₂Cl₂/MeOH (8/2),from which 2-(4- or5-Amino-2-hydroxy-benzoylamino)-4-thiocarbamoyl-butyric acid (6),compound of Formula XXI, was obtained (80% yield). MS (ESI), m/z 298(M⁺).

Example 10 General Synthetic Procedure of 4- or5-Amino-2-hydroxy-benzoic acid 4-thiocarbamoyl-phenyl ester (8)[Compound of Formula XXVII]

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1)

To the solution of 4- or 5-amino salicylic acid (10.0 mmol) in 25 mL ofdioxane and 12.5 mL of water, triethylamine (15.0 mmol) anddi-tert-butyl-dicarbonate (15.0 mmol) were added with stirring at 0° C.for ½ h. The reaction mixture was stirred mechanically for 24 h at roomtemperature. After evaporation of the solvent, 3M HCl (15 mL) was addeddrop wise to the residue. The precipitate is filtered, washed with waterand dried. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (9/1), from which 4- or5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1) was obtained (80%yield).

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-tert-butoxy-benzoic acid(2)

Compound (1) (12.0 mmol), conc. H₂SO₄ (6.0 mmol), and DCM (100 mL) werestirred under isobutylene gas (5 psi) for 6 h at rt. The solution waswashed with cold 10% NaHCO₃ (2×100 mL) and brine (100 mL), dried(Na₂SO₄) and evaporated. The residue was dissolved in 1:1 MeOH/CCl₄ (400mL), washed with water (300 mL), and then extracted with 1:1 MeOH/water(2×200 mL). The extract was dried (Na₂SO₄) and evaporated to a whitesolid (2), which was recrystallized by DCM/hexane (83% yield).

Synthesis of 4- or 5-Amino-2-hydroxy-benzoic acid 4-thiocarbamoyl-phenylester (8)

To the solution of 4- or 5-tert-butoxycarbonylamino-2-hydroxy-benzoicacid (2) (3.0 mmol) in 50 mL of dimethylformamide, hydroxybenzotriazole(3.3 mmol) and DCC (3.3 mmol) were added with stirring at 0° C. for 1 h.To the reaction mixture, 4-hydroxythiobenzamide (3.0 mmol) was added andstirred mechanically for 3 h at 0° C. and 72 h at room temperature.After filtration, the filtrate was evaporated under reduced pressure toremove the solvent. The oily residue thus obtained was dissolved inethyl acetate; the organic layer was washed with brine, dried onanhydrous MgSO₄, filtered and the solvent evaporated. The crudeintermediate (7) was treated with a solution of 40% TFA in CH₂Cl₂. After2 h the solvent was removed to obtain compound (8) as a crude residue.The residue was loaded on a silica gel open column and eluted withCH₂Cl₂/MeOH (8/2), from which 4- or 5-Amino-2-hydroxy-benzoic acid4-thiocarbamoylphenyl ester (8), compound of Formula XXVII, was obtained(48% yield).

Example 11 General Synthetic Procedure of 4- or5-Amino-2-hydroxy-benzoic acid4-[4-(4-hydroxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenylester (10) [Compound of Formula XVII]

Synthesis of (p-hydroxyphenyl)dithiophosphonic anhydride

Red P (4 g; 0.129 mol), S (4 g; 0.125 mol) and PhOH (4 g; 0.042 mol)were heated for 5.5 hr at 155-158° C.; the reaction mixture was cooledat room temperature and a precipitate was collected (5.5 g, 34% yield).m.p. 224-226° C. The NMR and MS analysis are consistent withp-hydroxyphenyl dithiophosphonic anhydride.

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1)

To the solution of 4- or 5-amino salicylic acid (10.0 mmol) in 25 mL ofdioxane and 12.5 mL of water, triethylamine (15.0 mmol) anddi-tert-butyl-dicarbonate (15.0 mmol) were added with stirring at 0° C.for ½ h. The reaction mixture was stirred mechanically for 24 h at roomtemperature. After evaporation of the solvent, 3M HCl (15 mL) was addeddrop wise to the residue. The precipitate is filtered, washed with waterand dried. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (9/1), from which 4- or5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1) was obtained (80%yield).

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-tert-butoxy-benzoic acid(2)

Compound (1) (12.0 mmol), conc. H₂SO₄ (6.0 mmol), and DCM (100 mL) werestirred under isobutylene gas (5 psi) for 6 h at rt. The solution waswashed with cold 10% NaHCO₃ (2×100 mL) and brine (100 mL), dried(Na₂SO₄) and evaporated. The residue was dissolved in 1:1 MeOH/CCl₄ (400mL), washed with water (300 mL), and then extracted with 1:1 MeOH/water(2×200 mL). The extract was dried (Na₂SO₄) and evaporated to a whitesolid (2), which was recrystallized by DCM/hexane (83% yield).

Synthesis of 4- or 5-Amino-2-hydroxy-benzoic acid4-[4-(4-hydroxy-phenyl)-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenylester (10)

To the solution of 4- or5-tert-butoxycarbonylamino-2-tert-butoxy-benzoic acid (2) (3.0 mmol) in50 mL of dimethylformamide, hydroxybenzotriazole (3.3 mmol) and DCC (3.3mmol) were added with stirring at 0° C. for 1 h. To the reactionmixture, p-hydroxyphenyldithiophosphonic anhydride (3.0 mmol) was addedand stirred mechanically for 3 h at 0° C. and 72 h at room temperature.After filtration, the filtrate was evaporated under reduced pressure toremove the solvent. The oily residue thus obtained was dissolved inethyl acetate; the organic layer was washed with brine, dried onanhydrous MgSO₄, filtered and the solvent evaporated. The crudeintermediate 9 was treated with a solution of TFA (40%) in CH₂Cl₂. After2 h the solvent was removed to obtain compound 10 as a crude residue.The residue was loaded on a silica gel open column and eluted withCH₂Cl₂/MeOH (8/2), from which 4- or 5-amino-2-hydroxy-benzoic acid4-[4-(4-hydroxy-phenyl-2,4-dithioxo-2λ⁵,4λ⁵-[1,3,2,4]dithiadiphosphetan-2-yl]-phenylester (10), compound of Formula XVII, was obtained (73% yield).

Synthesis of 4- or 5-amino-2-hydroxy-benzoic acidmercaptoethanesulfonate (2)

A mixture of sulfurous acid 2-mercapto-ethyl ester (0.1 mol) in 100 mlof ethyl acetate was added to the 4- or 5-aminosalicylic acid (1) (0.1mole in 100 ml of ethyl acetate) solution in 30-45 min at 20-25° C.under an inert atmosphere. Then the mixture was stirred at 0-5° C. for 1hour and filtered to give 4- or 5-amino-2-hydroxy-benzoic acidmercaptoethanesulfonate (2) (yield: 98%).

Example 12 Synthesis of 4 or5-amino-2-(2-Acetylamino-3-mercapto-propionyloxy)-benzoic acid (3)[Compound of Formula XII]

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1)

To the solution of 4- or 5-amino salicylic acid (10.0 mmol) in 25 mL ofdioxane and 12.5 mL of water, triethylamine (15.0 mmol) anddi-tertbutyl-dicarbonate (15.0 mmol) were added with stirring at 0° C.for ½ h. The reaction mixture was stirred mechanically for 24 h at roomtemperature. After evaporation of the solvent, 3 M HCl (15 mL) was addeddrop wise to the residue. The precipitate is filtered, washed with waterand dried. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (9/1), from which 4- or5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1) was obtained (80%yield).

Synthesis of 4 or5-amino-2-(2-Acetylamino-3-mercapto-propionyloxy)-benzoic acid (3)

To the solution of 2-acetylamino-3-mercapto-propionic acid (3.0 mmol) in50 mL of dimethylformamide, hydroxybenzotriazole (3.3 mmol) and DCC (3.3mmol) were added with stirring at 0° C. for 1 h. To the reactionmixture, 4- or 5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (2)(3.0 mmol) was added and stirred mechanically for 3 h at 0° C. and 72 hat room temperature. After filtration, the filtrate was evaporated underreduced pressure to remove the solvent. The oily residue thus obtainedwas dissolved in ethyl acetate; the organic layer was washed with brine,dried on anhydrous MgSO₄, filtered and the solvent evaporated. The crudeintermediate (2) was treated with a solution of TFA (40%) in CH₂Cl₂.After 2 h the solvent was removed to obtain compound (3) as a cruderesidue. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (8/2), from which 4 or5-amino-2-(2-Acetylamino-3-mercapto-propionyloxy)-benzoic acid (3),compound of Formula XII, was obtained (52% yield).

Example 13 Synthesis of 4 or 5-Amino-2-hydroxy-benzoic acid anhydridewith 2-Acetylamino-3-mercapto-propionic acid (4) [Compound of Formula X]

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1)

To the solution of 5-amino salicylic acid (10.0 mmol) in 25 mL ofdioxane and 12.5 mL of water, triethylamine (15.0 mmol) anddi-tertbutyl-dicarbonate (15.0 mmol) were added with stirring at 0° C.for ½ h. The reaction mixture was stirred mechanically for 24 h at roomtemperature. After evaporation of the solvent, 3M HCl (15 mL), was addeddrop wise to the residue. The precipitate is filtered, washed with waterand dried. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (9/1), from which 4- or5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1) was obtained (80%yield).

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-tert-butoxy-benzoic acid(2)

Compound (1) (12.0 mmol), conc. H₂SO₄ (6.0 mmol), and DCM (100 mL) werestirred under isobutylene gas (5 psi) for 6 h at rt. The solution waswashed with cold 10% NaHCO₃ (2×100 mL) and brine (100 mL), dried(Na₂SO₄) and evaporated. The residue was dissolved in 1:1 MeOH/CCl₄ (400mL), washed with water (300 mL), and then extracted with 1:1 MeOH/water(2×200 mL). The extract was dried (Na₂SO₄) and evaporated to a whitesolid (2), which was recrystallized by DCM/hexane (83% yield).

Synthesis of 4 or 5-Amino-2-hydroxy-benzoic acid anhydride with2-Acetylamino-3-mercapto-propionic acid (4)

To the solution of 4- or5-tert-butoxycarbonylamino-2-tert-butoxy-benzoic acid (2) (3.0 mmol) in50 mL of dimethylformamide, DCC (3.3 mmol) was added with stirring at 0°C. for 1 h. To the reaction mixture, 2-acetylamino-3-mercapto-propionicacid (3.0 mmol) was added and stirred mechanically for 3 h at 0° C. and72 h at room temperature. After filtration, the filtrate was evaporatedunder reduced pressure to remove the solvent. The oily residue thusobtained was dissolved in ethyl acetate; the organic layers were washedwith brine, dried on anhydrous MgSO₄, filtered and the solventevaporated. The crude intermediate (3) was treated with a solution ofTFA (40%) in CH₂Cl₂. After 2 h the solvent was removed to obtaincompound 4 as a crude residue. The residue was loaded on a silica gelopen column and eluted with CH₂Cl₂ MeOH (8/2), from which 4 or5-Amino-2-hydroxy-benzoic acid anhydride with2-Acetylamino-3-mercapto-propionic acid (4), compound of Formula X, wasobtained (68% yield).

Example 14 Synthesis of 4 or5-(2-Acetylamino-3-mercapto-propionylamino)-2-hydroxy-benzoic acid (5)[Compound of Formula XI]

Synthesis of 4 or5-(9H-Fluoren-9-ylmethoxycarbonylamino)-2-hydroxy-benzoic acid (1)

To the solution of 4- or 5-amino salicylic acid (10.0 mmol) in 25 mL ofdioxane and 12.5 mL of water, Na₂CO₃ 10% (15 mL) and Fmoc-OSu (15.0mmol) were added with stirring at 0° C. for 1/2 h. The reaction mixturewas stirred mechanically for 24 h at room temperature. After evaporationof the solvent, 3M HCl (15 mL) was added drop wise to the residue. Theprecipitate is filtered, washed with water and dried. The residue wasloaded on a silica gel open column and eluted with CH₂Cl₂/MeOH (9/1),from which 4- or 5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1)was obtained (90% yield).

Synthesis of 4 or 5-Amino-2-tert-butoxy-benzoic acid tert-butyl ester(3)

Compound (1) (12.0 mmol), conc. H₂SO₄ (6.0 mmol), and DCM (100 mL) werestirred under isobutylene gas (7 psi) for 24 h at room temperature. Thesolution was washed with cold 10% NaHCO₃ (2×100 mL) and brine (100 mL),dried (Na₂SO₄) and evaporated. The residue was dissolved in 1:1MeOH/CCl₄ (400 mL), washed with water (300 mL), and then extracted with1:1 MeOH/water (2×200 mL). The extract was dried (Na₂SO₄) and evaporatedto a white solid (2). The crude intermediate (2) was treated with asolution of diethylamine (33%) in THF. After 2 h the solvent was removedto obtain compound (3) as a crude residue. The residue was loaded on asilica gel open column and eluted with CH₂Cl₂/MeOH (8/2), from which 4or 5-Amino-2-tert-butoxy-benzoic acid tert-butyl ester (3) was obtained(67% yield).

Synthesis of 4 or5-(2-Acetylamino-3-mercapto-propionylamino)-2-hydroxy-benzoic acid (5)

To the solution of 2-acetylamino-3-mercapto-propionic acid (3.0 mmol) in50 mL of dimethylformamide, hydroxybenzotriazole (3.3 mmol) and DCC (3.3mmol) were added with stirring at 0° C. for 1 h. To the reactionmixture, 4 or 5-amino-2-tert-butoxy-benzoic acid tert-butyl ester (3)(3.0 mmol) was added and stirred mechanically for 3 h at 0° C. and 72 hat room temperature. After filtration, the filtrate was evaporated underreduced pressure to remove the solvent. The oily residue thus obtainedwas dissolved in ethyl acetate; the organic layer was washed with brine,dried on anhydrous MgSO₄, filtered and the solvent evaporated. The crudeintermediate 4 was treated with a solution of TFA (40%) in CH₂Cl₂. After2 h the solvent was removed to obtain compound (5) as a crude residue.The residue was loaded on a silica gel open column and eluted withCH₂Cl₂/MeOH (8/2), from which 4 or5-(2-Acetylamino-3-mercapto-propionylamino)-2-hydroxy-benzoic acid (5),compound of Formula XI, was obtained (78% yield).

Example 15 Synthesis of 4 or5-Amino-2-(2-mercapto-ethoxysulfonyloxy)-benzoic acid (3) [Compound ofFormula XXXIV]

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1)

To the solution of 4- or 5-amino salicylic acid (10.0 mmol) in 25 mL ofdioxane and 12.5 mL of water, triethylamine (15.0 mmol) anddi-tertbutyl-dicarbonate (15.0 mmol) were added with stirring at 0° C.for ½ h. The reaction mixture was stirred mechanically for 24 h at roomtemperature. After evaporation of the solvent, 3M HCl (15 mL) was addeddrop wise to the residue. The precipitate is filtered, washed with waterand dried. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (9/1), from which 4- or5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1) was obtained (80%yield).

Synthesis of: 4 or 5-Amino-2-(2-mercapto-ethoxysulfonyloxy)-benzoic acid(3)

To the solution of sulfuric acid mono-(2-mercapto-ethyl)ester (3.0 mmol)in 50 mL of dimethylformamide, hydroxybenzotriazole (3.3 mmol) and DCC(3.3 mmol) were added with stirring at 0° C. for 1 h. To the reactionmixture, 4- or 5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (2)(3.0 mmol) was added and stirred mechanically for 3 h at 0° C. and 72 hat room temperature. After filtration, the filtrate was evaporated underreduced pressure to remove the solvent. The oily residue thus obtainedwas dissolved in ethyl acetate; the organic layer was washed with brine,dried on anhydrous MgSO₄, filtered and the solvent evaporated. The crudeintermediate (2) was treated with a solution of TFA (40%) in CH₂Cl₂.After 2 h the solvent was removed to obtain compound 3 as a cruderesidue. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (8/2), from which 4 or5-Amino-2-(2-mercapto-ethoxysulfonyloxy)-benzoic acid (3), compound ofFormula XXXIV, was obtained (57% yield).

Example 16 Synthesis of 4 or 5-Amino-2-hydroxy-benzoic acid anhydridewith Sulfuric acid mono-(2-mercapto-ethyl)ester (4) [Compound of FormulaXXXIII]

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1)

To the solution of 5-amino salicylic acid (10.0 mmol) in 25 mL ofdioxane and 12.5 mL of water, triethylamine (15.0 mmol) anddi-tertbutyl-dicarbonate (15.0 mmol) were added with stirring at 0° C.for ½ h. The reaction mixture was stirred mechanically for 24 h at roomtemperature. After evaporation of the solvent, 3M HCl (15 mL) was addeddrop wise to the residue. The precipitate is filtered, washed with waterand dried. The residue was loaded on a silica gel open column and elutedwith CH₂Cl₂/MeOH (9/1), from which 4- or5-tert-Butoxycarbonylamino-2-hydroxy-benzoic acid (1) was obtained (80%yield).

Synthesis of 4- or 5-tert-Butoxycarbonylamino-2-tert-butoxy-benzoic acid(2)

Compound (1) (12.0 mmol), conc. H₂SO₄ (6.0 mmol), and DCM (100 mL) werestirred under isobutylene gas (5 psi) for 6 h at rt. The solution waswashed with cold 10% NaHCO₃ (2×100 mL) and brine (100 mL), dried(Na₂SO₄) and evaporated. The residue was dissolved in 1:1 MeOH/CCl₄ (400mL), washed with water (300 mL), and then extracted with 1:1 MeOH/water(2×200 mL). The extract was dried (Na₂SO₄) and evaporated to a whitesolid (2), which was recrystallized by DCM/hexane (83% yield).

Synthesis of 4 or 5-Amino-2-hydroxy-benzoic acid anhydride with Sulfuricacid mono-(2-mercapto-ethyl)ester (4)

To the solution of 4- or5-tert-Butoxycarbonylamino-2-tert-butoxy-benzoic acid (2) (3.0 mmol) in50 mL of dimethylformamide, DCC (3.3 mmol) was added with stirring at 0°C. for 1 h. To the reaction mixture, sulfuric acidmono-(2-mercapto-ethyl)ester (3.0 mmol) was added and stirredmechanically for 3 h at 0° C. and 72 h at room temperature. Afterfiltration, the filtrate was evaporated under reduced pressure to removethe solvent. The oily residue thus obtained was dissolved in ethylacetate; the organic layers were washed with brine, dried on anhydrousMgSO₄, filtered and the solvent evaporated. The crude intermediate (3)was treated with a solution of TFA (40%) in CH₂Cl₂. After 2 h thesolvent was removed to obtain compound (4) as a crude residue. Theresidue was loaded on a silica gel open column and eluted withCH₂Cl₂/MeOH (8/2), from which 4 or 5-Amino-2-hydroxy-benzoic acidanhydride with sulfuric acid mono-(2-mercapto-ethyl)ester (4), compoundof Formula XXXIII, was obtained (68% yield).

Characterization of Compounds Example 17 5-Amino-2-hydroxy-benzoic acid4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester [Referred to fromhereonin as Compound XXXV]

Thin layer chromatography was performed on Macherey-Nagel™ silica gel 50plates with fluorescent indicator and the plates were visualized with UVlight (254 nm). Kieselgel™ 60 was used for column chromatography. Allsynthetic reagents were purchased from the Aldrich-Sigma ChemicalCompany and were used without purification. Solvents were analyticalreagent grade or higher purity and were used as supplied. Solutions weredried over Na₂SO₄ and a Buchi™ R-114 rotavapor was utilized for theremoval of the solvents in vacuo. The structures were verifiedspectroscopically by proton ¹H-NMR and ¹³C-NMR. Spectra were recorded onVarian Mercury Plus 400 instrument. Spectra were recorded in DMSO. Thefollowing abbreviations are used to describe peak patterns whenappropriate: s (singlet), d (doublet). Chemical shifts are referred toMe₄Si as internal standard. Mass spectra of the synthesized productswere performed on Applayed Biosystem™ API 2000 mass spectrometry.Melting point was determined using a Kofler™ hot-stage apparatus and areuncorrected.

¹H NMR (DMSO) δ 7.07 (d, 2H), 7.38 (d, 2H), 7.46 (d, 2H), 7.79 (s, 1H),7.85 (s, 1H), 8.01 (d, 2H), 10.35 (s, —OH);

¹³C NMR (DMSO) δ 114.6; 119.6; 123.9; 127.7; 128.7; 129.4; 129.8; 136.1;153.8; 158.8; 165.4; 173.2; 189.7; 216.2

MS (EI), m/e 362 (M⁺);

m.p.: 93-95° C.

Example 18 5-Amino-2-hydroxy-benzoic acid 4-thiocarbamoyl-phenyl ester[Referred to hereonin as Compound XXVII]

Thin layer chromatography was performed on Macherey-Nagel silica gel 50plates with fluorescent indicator and the plates were visualized with UVlight (254 nm). Kieselgel 60 was used for column chromatography. Allsynthetic reagents were purchased from the Aldrich-Sigma ChemicalCompany and were used without purification. Solvents were analyticalreagent grade or higher purity and were used as supplied. Solutions weredried over Na₂SO₄ and a Buchi R-114 rotavapor was utilized for theremoval of the solvents in vacuo. The structures were verifiedspectroscopically by proton ¹H-NMR and ¹³C-NMR. Spectra were recorded onVarian Mercury Plus 400 instrument. Spectra were recorded in DMSO. Thefollowing abbreviations are used to describe peak patterns whenappropriate: s (singlet), d (doublet). Chemical shifts are referred toMe₄Si as internal standard. Mass spectra of the synthesized productswere performed on Applayed Biosystem API 2000 mass spectrometry. Meltingpoint was determined using a Kofler hot-stage apparatus and areuncorrected.

¹H NMR (DMSO): δ 7.03 (d, 1H), 7.31 (d, 2H), 7.32 (s, 1H), 7.71 (d, 1H),7.97 (d, 1H), 9.55 (s, NH₂), 9.91 (s, NH₂), 10.25 (s, —OH);

¹³C NMR (DMSO): 6114.4, 119.5, 122.1, 122.7, 129.2, 129.5, 138.1, 152.1,157.7, 165.9, 189.7, 199.7

MS (EI), m/e 289 (M⁺);

m.p.: 193-195° C.

Testing of Compounds Example 19 Dose-Ranging Study of2-hydroxy-5-amino-benzoic acid 4-(thioxo-5H-[1,2]dithiol-3-yl-phenylester hydrochloride (Compound XXXV) In TNBS-Induced Colitis in Mice

A standard experimental animal model of colitis induced by intracolonicadministration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to mice isused in the following example. A detailed description of this model hasbeen published (Santucci et al. (2003) Gastroenterology 124:1381-94) andis incorporated herein by reference. Briefly, 6-8 week old Balb/c micewere given TNBS intracolonically at a dose of 1.5 mg in 0.1 mL of 30%ethanol. The mice were randomized to the various treatment groups (n=6per group). Beginning one hour later and continuing every 12 h for 5days, the mice were treated orally with vehicle (1%carboxymethylcellulose (CMC)), 5-ASA (mesalamine) (25, 50 or 75 mg/kg)or with equimolar doses of 2-hydroxy-5-amino-benzoic acid4-(thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester hydrochloride (CompoundXXXV) (130 mg/kg), or with 66% (100 mg/kg), 50% (66 mg/kg) and 25% (33mg/kg) of that dose. The mice were evaluated (blindly) on the final dayof the study for the presence of diarrhea and fecal occult blood, andtheir body weights were measured. A “disease activity score” wascalculated based on these data (0 to 4 scale, as outlined in the papercited above). After sacrifice, a sample of the colon was excised formeasurement of myeloperoxidase (MPO) activity, as a marker ofgranulocyte infiltration. All results were compared to those obtainedwith healthy mice as well.

The results for the disease activity score and MPO activity are shown inFIG. 1 and FIG. 2, respectively. FIG. 1 shows that Compound XXXV wassuperior to mesalamine in reducing the activity score at equimolar dosesof 50 mg/kg and 75 mg/kg. Further, as shown in FIG. 2, MPO activity wassignificantly reduced (almost in half) at the highest doses tested.

Example 20 Comparison of Disease Activity Index and MPO Activity ofCompound XXXV with 5-ASA (Mesalamine) Alone, (ADT-OH) Alone, and aMixture of Mesalamine and ADT-OH

FIGS. 3 and 4 show the Disease Activity Index and MPO activity,respectively, using the same experimental animal model of colitis asdescribed above, in which Compound XXXV (130 mg/kg) was compared toequimolar doses of its two constituents, mesalamine (50 mg/kg) and5-p-hydroxyphenyl-1,2-dithione-3-thione (ADT-OH) (80 mg/kg), and amixture of mesalamine (50 mg/kg) and ADT-OH (80 mg/kg). *p<0.05 versusthe vehicle-treated group. Each group consisted of at least 5 rats.

FIG. 3 shows that Compound XXXV is almost twice as effective as eithermesalamine alone, ADT-OH alone or a mixture of mesalamine and ADT-OH inreducing disease symptoms. Further, FIG. 4 shows that Compound XXXVsignificantly reduced inflammation as indicated by the reduction ingranulocyte infiltration (reduced MPO activity).

Example 21 Comparison of Disease Activity Index and MPO Activity of5-Amino-2-(4-thiocarbamoyl-phenoxycarbonyloxy)-benzoic acid (CompoundXXVII)

FIGS. 6 and 7 show the Disease Activity Index and MPO, respectively,using the same experimental animal model of colitis as described above,in which Compound XXVII (100 mg/kg) was compared to equimolar doses ofits two constituents, mesalamine (50 mg/kg) and 4-hydroxythiobenzamide(4-HTB) (50 mg/kg), mesalamine alone (50 mg/kg) and 4-HTB alone (50mg/kg). *p<0.05 versus the vehicle-treated group. Each group consistedof at least 5 rats.

FIG. 5 shows that Compound XXVII is almost three times as effective aseither mesalamine alone, 4-HTB alone or a mixture of mesalamine and4-HTB in reducing disease symptoms. Further, FIG. 6 shows that CompoundXXVII significantly reduced inflammation as indicated by the reductionin granulocyte infiltration (reduced MPO activity).

Example 22 Effects of Mesalamine and Compound XXXV in TNBS-InducedColitis in Mice

The same model was used as described above. In this example, the effectsof mesalamine (50 mg/kg) were compared to those of equimolar doses ofCompound XXXV. In addition to measuring the severity of colitis bymeasuring disease activity score and MPO activity, tissues wereprocessed for measurement of a number of genes for inflammatorycytokines and other mediators.

In particular, mRNA expression in mice of tumour necrosis factor-alpha(TNF-α), interferon gamma (IFN-□γ), colonic interleukin (IL)-1, IL-2,IL-10, IL-12 p40, RANTES, cyclooxygenase (COX)-1, COX-2, constitutiveendothelial nitric oxide synthase (eNOS), and inducible NOS (iNOS) wasmeasured as described in Wallace et al. (1999) Gastroenterology 117:557-566, incorporated herein by reference.

Briefly, reverse transcription-polymerase chain reaction (RT-PCR) wasused to detect and quantify mRNA of the particularcytokine/chemokine/enzyme. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) was used as the “housekeeping gene” for mRNA expression (i.e.,as an internal control). For each sample, the ratio of the amplificationof the target gene to the amplification of GAPDH (expression of each ismeasured by performing densitometry on gels) was obtained. Comparisonswere then made between the relative amplification (expression) of thetarget gene in tissues for the treatment groups in comparison to theexpression in tissues from healthy controls. Thus, the data shown inFIGS. 7-14 represent the relative expression of the target gene(normalized to GAPDH expression) as a ratio to the expression in healthycontrols.

With reference to FIGS. 7-14, it is noteworthy that Compound XXXV wassuperior to mesalamine in every endpoint. It is particularly interestingthat Compound XXXV suppressed expression of mRNA for severalpro-inflammatory cytokines and chemokines that have been implicated inthe pathogenesis of inflammatory bowel disease. However, Compound XXXVdid not suppress expression of IL-10 mRNA, which is an anti-inflammatorycytokine.

In addition, Compound XXXV suppressed both COX-1 and COX-2 mRNA. COX-1and COX-2 are involved in the synthesis of prostaglandins, which areimportant in inflammation. Further, Compound XXXV also suppressed eNOSand iNOS mRNA. Both eNOS and iNOS have been implicated in diseases ofthe GI tract

Example 23 Comparison of the Effects of Compound XXXV Versus Mesalaminein Inhibiting Viability of HT-29 Human Colon Cancer Cells In Vitro

HT-29 cells were grown in culture using standard methods. The cells wereexposed to vehicle (DMSO), mesalamine or Compound XXXV. Concentrationsranging from 0.1 to 10 μM were tested, with each concentration tested in6 wells. At the end of 72 h of exposure to the test drugs, cellviability was measured using the MTT[3-(4,5-dimethylthiaxol-2-yl)-2,5-diphenyltetrazolium bromide] assay(Carmichael et al. (1978) Cancer Res. 47, 936-942), incorporated hereinby reference. Cell viability rates were calculated as a percent of thevehicle (DMSO)-treated cells and results are given in Table 1.

TABLE 1 Concentrations (μM) Drug 0.1 1 10 Mesalamine 103.8 ± 2.5 101.2 ±3.5 91.1 ± 3.2 XXXV  88.4 ± 2.9**  87.3 ± 2.3** 79.6 ± 1.9** **p < 0.01versus the mesalamine-treated group (same concentration)

Example 24 Comparison of the Effects of Compound XXXV versus Mesalaminein a Rat Model of Visceral Pain Perception

A rat model of visceral pain perception, a pre-clinical model ofirritable bowel syndrome, was used in the following example. Rats (male,Wistar, 200-250 g, obtained from Charles River, Monza, Italy), werehoused in plastic cages and maintained under controlled conditions with12-hours light/dark cycle with lights on at 7.00 AM. Tap water andstandard laboratory chow were freely available. Before experiments, ratswere individually trained by spending 2-3 hours per day in a plexiglasscage for 2-3 days. It allowed them to adjust to a movement-restrictionenvironment. Food was withheld for 12 hours before colorectal distension(CRD) recording were performed. Experiments were performed in awake ratsand were conducted in a blind manner in that the observer was not awareof the identity of drug administered to each animal.

In the testing day, rats were sedated with ether inhalation and a 2 cmlong latex balloon was inserted intrarectally 2 cm from the anal vergeand fixed at the base of the tail. The balloon was connected via adouble-barreled cannula to a pressure transducer to continuouslymonitoring the rectal pressure by a computer (PowerLab PC, A.D.Instruments, Milford, Mass., USA) and to a syringe forinflation/deflation of the balloon. The rats were then housed in a smallcage (20×8×8 cm) on an elevated Plexiglas™ platform and allowed to wakeup and adapt for 1 hour. After recovery from sedation, animals underwentthe CRD procedure and behavioral responses were tested. The night beforethe experiments, the balloons were inflated and left overnight so thelatex stretched and the balloons became compliant.

CRD of 20 seconds, performed every 5 minutes, was applied in incrementof 0.4 ml starting from 0.4 ml up to 1.6 ml water. To achieve anaccurate measurement of the colonic parameters and perception, thedistensions were repeated twice for each intensity and data for eachanimal were averaged for analysis. Each animal underwent a double set ofCRD. Twenty minutes after the first sequence of CRD (0.4 mL-1.6 mlwater), drugs were administered intraperitoneally (i.p.) and a secondset of CRD was performed. Behavioral responses during the first and thesecond set of CRD were assessed and compared.

Behavioral response to CRD was assessed by measuring the abdominalwithdrawal reflex (AWR) using a semiquantitative score (1). The AWR isan involuntary motor reflex similar to the visceromotor reflex, but ithas the great advantage that, in contrast to the latter, it does notrequire abdominal surgery to implant recording electrodes and wires inthe abdominal muscle wall which may cause additional sensitization (seeNess, T. J. and Gebhart, G. F. (1990) Pain 41:167-234, incorporatedherein by reference).

Measurement of the AWR consisted of visual observation of the animalresponse to graded CRD by blinded observer and assignment of an AWRscore according with the behavioral scale as previously described inAl-Chaer, E. D. et al. (2000) Gastroenterology 19: 1276-85, incorporatedherein by reference, in which grade 0 corresponds to no behavioralresponse to CRD, grade 1 corresponds to brief head movement at the onsetof the stimulus followed by immobility, grade 2 corresponds to a mildcontraction of abdominal muscles although the rats does not lift theabdomen off the platform, grade 3 corresponds to a strong contraction ofthe abdominal muscles with the lifting of the abdomen off the platform,and grade 4 corresponds to a severe contraction of the abdominal musclemanifested by body arching and the lifting of the abdomen and of thepelvic structures and scrotum.

The effects of mesalamine and Compound XXXV on colonic compliance andsensitivity were determined using a total of 8 fasting rats. Toinvestigate whether the administration of mesalamine and Compound XXXVcould revert pain induced by CRD, after the first sequence of CRD, 4rats were treated with mesalamine at the dose of 100 mg/kg i.p. orCompound XXXV at the dose of 100 mg/kg i.p., after which a second set ofCRD was repeated. Results from these experiments are shown in FIGS. 15(a) and (b).

To determine the effect of mesalamine or Compound XXXV on colonic smoothmuscle, the compliance of the colo-rectum during CRD was obtained fromintracolo-rectal volume and pressure and expressed as mL/mmHg. Theseresults are shown in FIGS. 16( a) and (b).

All data are presented as the mean±SEM, with sample sizes of 4rats/group; statistical comparison of paired data was performed by theWilcoxon signed rank test. An associated probability (p value) of lessthat 5% was considered significant.

FIGS. 15( a) and (b) show that Compound XXXV is more effective thanmesalamine (and vehicle) in reducing visceral pain in response tocolorectal distension. Further, Compound XXXV successfully reducedintrarectal pressure, as shown in FIG. 16( b).

Thus, Compound XXXV, which has also been shown to have effectiveanti-inflammatory activity, is useful in treating various inflammatoryconditions of the alimentary tract, as well as functionalgastrointestinal disorders such as irritable bowel syndrome, dyspepsia,etc., that are characterized by increased visceral nociception (with orwithout accompanying inflammation).

Example 25 Compound XXXV and Compound XXVII Pain Perception Scores withor without Glibenclamide

A rat model of visceral pain perception as described above was used tocompare pain perception scores for both Compound XXXV and Compound XXVIIwith or without glibenclamide, an inhibitor of ATP-sensitive K⁺(K_(ATP)) channels.

FIG. 17 shows the pain perception score in response to 0.8 mL ofcolorectal distention in groups of rats (at least 5 per group) treatedwith vehicle, mesalamine (100 mg/kg), Compound XXXV (100 mg/kg) orCompound XXVII (100 mg/kg). Both Compound XXXV and XXVII significantlyreduced the pain perception (*p<0.05 versus the vehicle-treated group),while mesalamine had no significant effect. The reduction of painperception by Compound XXXV and Compound XXVII was reversed bypretreatment with glibenclamide (10 mg/kg i.p. 30 min before), whileglibenclamide pretreatment did not affect the pain perception in thegroups treated with vehicle or mesalamine, suggesting that theanti-nociceptive activity of Compounds XXXV and XXVII may be mediated byATP-sensitive K⁺ (K_(ATP)) channels.

Example 26 Comparison of Compound XXXV and Compound XXVII PainPerception Scores with Mesalamine

FIG. 19 shows results from an experiment using the same pain model asdescribed above. The effects of treatment with mesalamine (50 mg/kg)were compared to the effects of equimolar doses Compound XXXV (130mg/kg), ADT-OH (80 mg/kg), Compound XXVII (100 mg/kg) and 4-HTB (50mg/kg). Only Compound XXXV and Compound XXVII significantly reduced painperception (*p<0.05) when compared to the vehicle-treated group.

Example 27 Effects of Compounds XXXV and XXVII on Leukocyte Adherence tothe Vascular Endothelium In Vivo

Leukocyte adherence was studied using intravital microscopy, asdescribed in detail previously (Wallace et al., (1993) Am. J. Physiol.265: 993-998, incorporated hereto by reference). Rats were anesthetizedwith pentobarbital sodium (60 mg/kg i.p.) and cautery incisions weremade along the abdominal region. A tracheotomy was performed tofacilitate breathing. The rats were placed in a supine position, and asegment of the mesentery was exteriorized through the abdominalincision. The mesentery was carefully placed over an optically clearviewing pedestal that allowed for trans-illumination of a 2-cm² segmentof tissue. All exposed tissue was covered with saline-soaked gauze tominimize dehydration. The temperature of the pedestal was kept at 37° C.and the mesentery was superfused with warmed bicarbonate-buffered saline(pH 7.4). An intravital microscope (Nikon L25/0.35) and a ×10 eyepiecewere used to observe the mesenteric microcirculation. Post-capillaryvenules with diameters ranging from 20 to 40 μm were selected for thestudy. A video camera mounted on the microscope (Panasonic™ digital5000) projected the image onto a monitor, and the images were recordedfor playback analysis using a videocassette recorder. Images of themesenteric microcirculation were recorded 5 minutes prior to aspirinadministration (baseline), at the time of aspirin administration (time0-5) and every 15 minutes for 60 minutes. Leukocyte adherence wasblindly quantified from videotaped images of the vessels made over 5-minperiods as the number of leukocytes that remained stationary along thevessel wall for 30 s or more (expressed per 100 μm venule length).Groups of rats (at least 5 in each) were pretreated with Compound XXXV(130 mg/kg), Compound XXVII (100 mg/kg), mesalamine (50 mg/kg), orvehicle 60 min prior to aspirin (or vehicle) administration. These drugswere given intragastrically. In some experiments, rats were treated withglibenclamide (10 mg/kg i.p.) or vehicle 30 min prior to administrationof these compounds.

FIG. 19 shows leukocyte adherence in response to intragastricadministration of aspirin, and the effects of the compounds. Aspirinmarkedly increased leukocyte adherence over that observed in thebaseline period (*p<0.05 versus the vehicle+vehicle group. Pretreatmentwith compound XXXV, but not with mesalamine, prevented theaspirin-induced increase in leukocyte adherence. Glibenclamide alone didnot affect leukocyte adherence, and did not affect the magnitude ofaspirin-induced leukocyte adherence. Glibenclamide also had no effect inthe group treated with mesalamine plus aspirin. However, glibenclamidereversed the inhibitory effect of Compound XXXV on aspirin-inducedleukocyte adherence.

FIG. 20 shows the leukocyte adherence for the final time period of theexperiment (minutes 60-65). This graph illustrates the ability ofCompound XXXV and Compound XXVII to suppress aspirin-induced leukocyteadherence, and the ability of glibenclamide pretreatment to reverse thisinhibitor effect on leukocyte adherence.

Example 28 Generation of H₂S by 5-Amino-2-hydroxy-benzoic acid4-(5-thioxo-5H-[1,2]dithiol-3-yl)-phenyl ester (Compound XXXV) and 4- or5-Amino-2-(4-thiocarbamoyl-phenoxycarbonyloxy)-benzoic acid (referred toas Compound XXVII)

Two compounds were tested, Compound XXXV and Compound XXVII, for H₂Sgeneration under three different conditions. Concentrations of H₂S thatwere generated within 1 hour from 1 mM concentrations of L-cysteine, theH₂S-releasing moiety of Compound XXXV, ADT-OH(5-(4-Amino-phenyl)-[1,2]dithiole-3-thione, the H₂S releasing moiety ofCompound XXVII, 4-HBT (4-hydroxythiobenzamide) were also measured. H₂Srelease was tested under three conditions: (i) when the compound was inbuffer, (ii) when the compound was in a liver homogenate, and (iii) whenthe compound was in the liver homogenate together with an inhibitor ofcystathionine γ-lyase (PAG=DL-propargylglycine; 2 mM). Results are shownin FIG. 13. *p<0.05 compared to the release from the vehicle group.^(ψ)p>0.05 versus the corresponding ‘homogenate’ group. The enzymaticcapacity for H₂S production was determined using the same reactor asdescribed previously (Khan et al. (1980) Microchem J. 25: 388-395,incorporated herein by reference). Two ml of an assay reaction mixturewas introduced in the reactor. The mixture contained 1 mM L-cysteine (orcompound), 2 mM pyridoxal 5′-phosphate, 100 mM potassium phosphatebuffer (pH=7.4). A constant stream of nitrogen was passed through themixture via gas-inlet capillary. Reactions were initiated bytransferring the tubes from ice bath to a 37° C. water bath. The streamof nitrogen carried the sulfide acid in the second reactor containing 4ml of sulfide anti-oxidant buffer (SAOB) solution, consisting of 2M KOH,1M salicylic acid and 0.22M ascorbic acid at pH 12.8[5]. Afterincubating at 37° C. for 90 minutes, 1 ml of 10% trichloroacetic acidsolution was added to mixture to stop the reaction. The remainder H₂S inthe mixture was carried out via nitrogen stream by other 60 minutes ofincubation at 37° C. The concentration of sulfide in SAOB solution wasmeasured with a sulfide sensitive electrode (Model 9616 S²⁻/Ag⁺electrode, Orion Research, Beverly, Mass., USA. For studies in which thetest compounds were incubated in liver homogenate, 100-150 mg ofisolated rat livers were homogenized in 1 ml of ice-cold T-PER proteinextractor. The homogenates were added to the reaction mixture at aconcentration of 10% (wt/vol). DL-propargylglycine 2 mM was incubatedwith liver homogenates for 5 min at 37° C. prior the enzyme reaction.Khan, S. U. Morris, G. F. and Hidiroglou, M. (1980) Rapid estimation ofsulfide in rumen and blood with a sulfide-specific ion electrode.Microchem J. 25:388-395, incorporated herein by reference.

The results shown in FIG. 21 suggest that 4- or 5-ASA derivatives of thepresent invention and, in particular, Compound XXXV and XXVII, have thefollowing distinct features:

-   -   1. The derivatives release H₂S spontaneously (in buffer), which        is desirable for a topical effect in the gut. The H₂S-releasing        moieties alone, ADT-OH and 4-HTB, and L-cysteine did not release        significant H₂S when incubated only in buffer;    -   2. The release of H₂S is greater when in the presence of tissue;    -   3. The release of H₂S from 4- or 5-ASA derivatives (other than        4- or 5-amino-2-hydroxy-benzoic acid anhydride with N-acetyl        cysteine (Formula X), occurs independent of the activity of the        two main enzymes for endogenous synthesis of H₂S (cystathionine        β-synthase and cystathionine-γ-lyase). This is demonstrated by        lack of effect of an inhibitor of those enzymes (PAG;        DL-propargylglycine), on H₂S generation from Compound XXXV and        Compound XXVII. In contrast, the release of H₂S from L-cysteine        is markedly inhibited by PAG;    -   4. The concentrations of H₂S produced from Compound XXXV and        Compound XXVII are in the 10-20 uM range when 1 mM of the        compound was used. Concentrations of up to 5 mM mesalamine can        be measured in the colonic lumen after patients have taken the        usual doses of this drug (Dig. Dis. Sci. 1989; 34: 573-578).        Endogenous concentrations of H₂S can be as much as 160 μM        (Antioxid. Redox Signal. 2003; 5, 493-501). Both Compound XXXV        and XXVII release H₂S at concentrations within the physiological        range thereby minimizing the chances of H₂S-related toxicity. It        is understood, however, that when n-acetylcysteine is the H₂S        releasing moiety (compounds of Formula X), a lower dose will be        used due to the greater release of H₂S from cysteine.

Example 29 Vasorelaxant Effects of H₂S Releasing Moieties

The following experiments were performed essentially using the protocolas disclosed in Bucci, M. et al. (2004) Diabetic mouse angiopathy islinked to progressive sympathetic receptor deletion coupled to anenhanced caveolin-1 expression. Arterioscler Thromb Vasc Biol 24:721-726, incorporated herein by reference. CD-1 mice were sacrificed andthoracic aorta was rapidly dissected and cleaned from fat and connectivetissue. Rings of 1.5-2 mm length were cut and mounted on isolated organbath (Fort 10 World Precision Instruments, USA) filled with gassed Krebssolution (95% O2+5% CO₂) at 37° C. Changes in isometric tension wererecorded with PowerLab™ data acquisition system (Ugo Basile, Italy). Thecomposition of the Krebs solution was as follows (mol/l): NaCl 0.118,KCl 0.0047, MgCl2 0.0012, KH2PO4 0.0012, CaCl2 0.0025, NaHCO3 0.025 andglucose 0.010. Rings were initially stretched until a resting tension of1.5 g was reached and allowed to equilibrate for at least 40 minutesduring which tension was adjusted, when necessary, to 1.5 g and bathingsolution was periodically changed. In a preliminary study a restingtension of 1.5 g was found to develop the optimal tension to stimulationwith contracting agents.

In each experiment rings were standardized using L-phenylephrine (PE) 1μmol/l until the responses were reproducible. To evaluate thevasorelaxant effect of tested compounds, cumulativeconcentration-response curves were performed (10 nM-3 mM) on PE (1 μM)precontracted rings for the following compounds:

2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide(Lawesson's Reagent), thioacetamide, NaHS, 4-HTB and Na₂S. Vehiclerepresents buffer but no compound. Curves to tested compounds wereconstructed in presence of endothelium. To assess endothelium integritycumulative concentration-response curve to Ach (10 nM-30 μM) wasperformed on PE precontracted rings.

Data obtained are shown in FIG. 22 and are expressed as mean±SEM. Thelevel of statistical significance was determined by 2-way analysis ofvariance (ANOVA) followed by Bonferroni's test for multiple comparison,using the GraphPad™ software.

FIG. 22 shows that the H2S releasing moieties of the present invention,namely, Lawesson's Reagent, 4-HTB and thioacetamide, all showedsignificant vasorelaxant effects, which were concentration dependent,when compared to vehicle. Further, the % relaxation curves were allcomparable to those obtained when using NaHS and Na₂S.

1. A method of treating an inflammatory condition of thegastrointestinal tract in a subject in need of such treatment, saidmethod comprising administering to the subject an amount effective totreat the inflammatory condition of the gastrointestinal tract of acompound of the general formula:A-L-R  (I) where: A is

where —N═ is either at position 4 or 5,

where —NH is either at position 4 or 5,

where —NH₂ is either at position 4 or 5, or

where —NH₂ is either at position 4 or 5; L is either O, O—C═O, S, N or acovalent bond, to form an ester linkage, an anhydride linkage, athioester linkage, an amide linkage or an azo linkage; and R is ahydrogen sulfide releasing moiety selected from the group consisting of:

or a pharmacologically acceptable salt thereof.
 2. The method accordingto claim 1 wherein the compound is 4- or 5-amino-2-hydroxy-benzoic acidanhydride with N-acetyl cysteine, or a pharmacologically acceptable saltthereof.
 3. The method according to claim 1 wherein the inflammatorycondition of the gastrointestinal tract is Crohn's disease.
 4. Themethod according to claim 1 wherein the inflammatory condition of thegastrointestinal tract is ulcerative colitis.
 5. The method according toclaim 1 wherein the inflammatory condition of the gastrointestinal tractis irritable bowel syndrome.
 6. A method for preventing colon cancer ina subject, said method comprising administering to the subject an amounteffective of a compound of the general formula:A-L-R  (I) where: A is

where —N═ is either at position 4 or 5,

where —NH is either at position 4 or 5,

where —NH₂ is either at position 4 or 5, or

where —NH₂ is either at position 4 or 5; L is either O, O—C═O, S, N or acovalent bond, to form an ester linkage, an anhydride linkage, athioester linkage, an amide linkage or an azo linkage; and R is ahydrogen sulfide releasing moiety selected from the group consisting of:

or a pharmacologically acceptable salt thereof.
 7. The method accordingto claim 6 wherein the compound is 4- or 5-amino-2-hydroxy-benzoic acidanhydride with N-acetyl cysteine, or a pharmacologically acceptable saltthereof.